Uses of kappa opioid synthetic peptide amides

ABSTRACT

The invention relates to methods of use of synthetic peptide amides that are ligands of the kappa opioid receptor in the treatment and prevention of kappa opioid receptor-associated diseases and conditions; and particularly to uses of these agonists in the prophylaxis, inhibition and treatment of pain, inflammation and pruritis associated with a variety of diseases, disorders and conditions. Inflammatory conditions preventable or treatable by the methods of the invention include diseases and conditions associated with elevated levels of a proinflammatory cytokines, such as TNF-α, IL-β, IL-6, MMP-1 and MMP-3. Such diseases and conditions include cardiovascular inflammation, neurological inflammation, skeletal inflammation, muscular inflammation, gastrointestinal inflammation, ocular inflammation, otic inflammation, inflammation due to insect bites and inflammation due to wound healing; atherosclerosis, ischemia, restenosis and vasculitis; of asthma, Sjogren&#39;s syndrome, pulmonary inflammation, chronic airway inflammation and chronic obstructive pulmonary disease (COPD), allergy, psoriasis, psoriatic arthritis, eczema, scleroderma, atopic dermatitis and systemic lupus erythematosus, arthritis, synovitis, osteomyelitis, rheumatoid arthritis, osteoarthritis and ankylosing spondylitis; septicemia and septic shock, diabetes, glucose intolerance, insulin resistance and obesity, colitis, ulcerative colitis, Crohn&#39;s disease, IBD and IBS, and the inflammatory diseases and conditions due to tumor proliferation, tumor metastasis or transplantation rejection.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.12/480,059, filed Jun. 8, 2009 which is a is a continuation-in-part ofU.S. application Ser. No. 12/119,311 filed May 12, 2008, now U.S. Pat.No. 7,713,937, which is a continuation-in-part of U.S. application Ser.No. 11/938,771 filed Nov. 12, 2007, now U.S. Pat. No. 7,402,564 whichclaims priority to U.S. provisional applications, Ser. Nos. 60/858,109filed Nov. 10, 2006, and 60/928,550 filed May 10, 2007; and is acontinuation-in-part of U.S. Ser. No. 11/938,776 filed Nov. 12, 2007which claims priority to U.S. provisional applications, Ser. Nos.60/858,120; 60/858,121 and 60/858,123 filed Nov. 10, 2006, and60/928,527, 60/928,551, and 60/928,557 filed May 10, 2007, all of whichare expressly incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to synthetic peptide amides incorporating D-aminoacids in the peptide chain and more particularly to such syntheticpeptide amides that are kappa opioid receptor agonists, and methods fortheir use as prophylactic and therapeutic agents for the prevention,inhibition and treatment of kappa opioid receptor associated diseasesand conditions, including pain, inflammation and pruritis.

BACKGROUND

Kappa opioid receptors have been suggested as targets for interventionfor treatment or prevention of a wide array of diseases and conditionsby administration of kappa opioid receptor agonists. See for example,Jolivalt et al., Diabetologia, 49(11):2775-85; Epub Aug. 19, 2006),describing efficacy of asimadoline, a kappa receptor agonist in rodentdiabetic neuropathy; and Bileviciute-Ljungar et al., Eur. J. Pharm.494:139-46 (2004) describing the efficacy of kappa agonist U-50,488 inthe rat chronic constriction injury (CCI) model of neuropathic pain andthe blocking of its effects by the opioid antagonist, naloxone. Theseobservations support the use of kappa opioid receptor agonists fortreatment of diabetic, viral and chemotherapy-induced neuropathic pain.The use of kappa receptor agonists for treatment or prevention ofvisceral pain including gynecological conditions such as dysmenorrhealcramps and endometriosis has also been reviewed. See for instance,Riviere, Br. J. Pharmacol. 141:1331-4 (2004).

Kappa opioid receptor agonists have also been proposed for the treatmentof pain, including hyperalgesia. Hyperalgesia is believed to be causedby changes in the milieu of the peripheral sensory terminal occursecondary to local tissue damage. Tissue damage (e.g., abrasions, burns)and inflammation can produce significant increases in the excitabilityof polymodal nociceptors (C fibers) and high threshold mechanoreceptors(Handwerker et al. (1991) Proceeding of the VIth World Congress on Pain,Bond et al., eds., Elsevier Science Publishers BV, pp. 59-70; Schaibleet al. (1993) Pain 55:5-54). This increased excitability and exaggeratedresponses of sensory afferents is believed to underlie hyperalgesia,where the pain response is the result of an exaggerated response to astimulus. The importance of hyperalgesia in the post-injury pain statehas been repeatedly demonstrated and appears to account for a majorproportion of pain experienced in the post-injury/inflammatory state.See for example, Woold et al. (1993) Anesthesia and Analgesia 77:362-79;Dubner et al. (1994) In, Textbook of Pain, Melzack et al., eds.,Churchill-Livingstone, London, pp. 225-242.

Kappa opioid receptors have been suggested as targets for the preventionand treatment of cardiovascular disease. See for example, Wu et al.“Cardioprotection of Preconditioning by Metabolic Inhibition in the RatVentricular Myocyte—Involvement of kappa Opioid Receptor” (1999)Circulation Res vol. 84: pp. 1388-1395. See also Yu et al.“Anti-Arrhythmic Effect of Kappa Opioid Receptor Stimulation in thePerfused Rat Heart: Involvement of a cAMP-Dependent Pathway” (1999) JMol Cell Cardiol. vol. 31(10): pp. 1809-1819.

It has also been found that development or progression of these diseasesand conditions involving neurodegeneration or neuronal cell death can beprevented, or at least slowed, by treatment with kappa opioid receptoragonists. This improved outcome is believed to be due to neuroprotectionby the kappa opioid receptor agonists. See for instance, Kaushik et al.“Neuroprotection in Glaucoma” (2003) J. Postgraduate Medicine vol. 49(1): pp. 90-95.

The presence of kappa opioid receptors on immune cells (Bidlak et al.,(2000) Clin. Diag. Lab. Immunol. 7(5):719-723) has been implicated inthe inhibitory action of a kappa opioid receptor agonist, which has beenshown to suppress HIV-1 expression. See Peterson P K et al., BiochemPharmacol. 2001, 61(19):1145-51.

Walker, Adv. Exp. Med. Biol. 521:148-60 (2003) appraised theanti-inflammatory properties of kappa agonists for treatment ofosteoarthritis, rheumatoid arthritis, inflammatory bowel disease andeczema. Bileviciute-Ljungar et al., Rheumatology 45:295-302 (2006)describe the reduction of pain and degeneration in Freund'sadjuvant-induced arthritis by the kappa agonist U-50,488.

Wikstrom et al., J. Am. Soc. Nephrol. 16:3742-7 (2005) describes the useof the kappa agonist, TRK-820 for treatment of uremic and opiate-inducedpruritis, and Ko et al., J. Pharmacol. Exp. Ther. 305:173-9 (2003)describe the efficacy of U-50,488 in morphine-induced pruritis in themonkey.

Application of peripheral opioids including kappa agonists for treatmentof gastrointestinal diseases has also been extensively reviewed. See forexample, Lembo, Diges. Dis. 24:91-8 (2006) for a discussion of use ofopioids in treatment of digestive disorders, including irritable bowelsyndrome (IBS), ileus, and functional dyspepsia.

Ophthalmic disorders, including ocular inflammation and glaucoma havealso been shown to be addressable by kappa opioids. See Potter et al.,J. Pharmacol. Exp. Ther. 309:548-53 (2004), describing the role of thepotent kappa opioid receptor agonist, bremazocine, in reduction ofintraocular pressure and blocking of this effect by norbinaltorphimine(norBNI), the prototypical kappa opioid receptor antagonist; andDortch-Carnes et al., CNS Drug Rev. 11(2):195-212 (2005). U.S. Pat. No.6,191,126 to Gamache discloses the use of kappa opioid agonists to treatocular pain. Otic pain has also been shown to be treatable byadministration of kappa opioid agonists. See U.S. Pat. No. 6,174,878also to Gamache.

Kappa opioid agonists increase the renal excretion of water and decreaseurinary sodium excretion (i.e., produces a selective water diuresis,also referred to as aquaresis). Many, but not all, investigatorsattribute this effect to a suppression of vasopressin secretion from thepituitary. Studies comparing centrally acting and purportedlyperipherally selective kappa opioids have led to the conclusion thatkappa opioid receptors within the blood-brain barrier are responsiblefor mediating this effect. Other investigators have proposed to treathyponatremia with nociceptin peptides or charged peptide conjugates thatact peripherally at the nociceptin receptor, which is related to butdistinct from the kappa opioid receptor (D. R. Kapusta, Life Sci.,60:15-21, 1997) (U.S. Pat. No. 5,840,696). U.S. Pat Appl. 20060052284.

SUMMARY OF THE INVENTION

The invention provides a method of prophylaxis or treatment orinhibition of a kappa opioid receptor-associated disease or condition,including pain, inflammation and pruritis, in a mammal, wherein themethod includes administering a composition comprising an effectiveamount of a synthetic peptide amide of the formula I to the mammal. Thestructure of formula I is as follows.

Also useful in the methods of the present invention are stereoisomers,prodrugs, pharmaceutically acceptable salts, hydrates, solvates, acidsalt hydrates, N-oxides and isomorphic crystalline forms of thesynthetic peptide amides of formula I.

The invention also provides uses of the synthetic peptide amides for thepreparation of medicaments and pharmaceutical compositions useful forthe prevention or treatment of a kappa opioid receptor-associateddisease or condition in a mammal

The kappa opioid receptor-associated disease or condition preventable ortreatable by the methods of the present invention include inflammatorydiseases and conditions.

The invention also provides a method of treating or preventinghyponatremia or hypokalemia, and thereby treating or preventing adisease or condition associated with hyponatremia or hypokalemia, suchas congestive heart failure, liver cirrhosis, nephrotic syndrome,hypertension, or edema, and preferably where increased vasopressinsecretion is associated with said disease or condition, wherein themethod includes administering to a mammal an aquaretically effectiveamount of a synthetic peptide amide of the invention in apharmaceutically acceptable diluent, excipient or carrier.

In formula I,

each Xaa₁ is independently chosen from the following D-amino acids:(A)(A′)D-phenylalanine, (A)(A′)α-methyl-D-phenylalanine, D-tyrosine,D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, D-neopentylglycine,D-phenylglycine, D-homo-phenylalanine, β-(E)D-alanine and D-tert-Leu,wherein each (A) and each (A′) are phenyl ring substituentsindependently chosen from —H, —F, —Cl, —NO₂, —CH₃, —CF₃, —CN, —CONH₂,and wherein each (E) is independently chosen from tert-butyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, furyl, pyridyl,thienyl, thiazolyl and benzothienyl.

Each Xaa₂ is independently chosen from (A)(A′)D phenylalanine,3,4-dichloro-D-phenylalanine, (A)(A′)(α-Me)D-phenylalanine,D-1-naphthylalanine, D-2-naphthylalanine, D-tyrosine, (E)D-alanine, andD-tryptophan, wherein (A), (A′) and (E) are defined above.

Each Xaa₃ is independently chosen from D-norleucine, D-phenylalanine,(E)-D-alanine, D-leucine, (α-Me)D-leucine, D-isoleucine,D-allo-isoleucine, D-homoleucine, D-valine, and D-methionine.

Each Xaa₄ is independently chosen from (B)₂D-arginine,(B)₂D-norarginine, (B)₂D-homoarginine, ζ-(B)D-homolysine,D-2,3-diaminopropionic acid, ε-(B)D-lysine, ε-(B)₂-D-lysine,D-aminomethylphenylalanine, amidino-D-aminomethyl-phenylalanine,γ-(B)₂D-α,γ-diaminobutyric acid, ζ-(B)₂α-(B′)D-ornithine,D-2-amino-3(4-piperidyl)-propionic acid,D-2-amino-3(2-aminopyrrolidyl)propionic acid,D-α-amino-β-amidino-propionic acid, α-amino-4-piperidineacetic acid,cis-α,4-diaminocyclo-hexane acetic acid,trans-α,4-diaminocyclohexaneacetic acid,cis-α-amino-4-methyl-aminocyclo-hexane acetic acid,trans-α-amino-4-methylaminocyclohexane acetic acid,α-amino-1-amidino-4-piperidineacetic acid,cis-α-amino-4-guanidino-cyclohexane acetic acid, andtrans-α-amino-4-guanidinocyclohexane acetic acid, wherein each (B) isindependently chosen from —H and C₁-C₄ alkyl, and (B′) is —H or (α-Me);and p is zero or 1, such that when p is 1 G is bonded to Xaa₄ and when pis zero, then G is directly bonded to Xaa₃.

The moiety G is selected from one of the following moieties:

Either (i) G is

wherein p, q, r, s and t are each independently zero or 1, provided thatat least one of s and t is 1, such that when t is 1 L is bonded to Xaa₄and when t is zero, then L is directly bonded to Xaa₃. The moiety L is alinker chosen from ε-D-Lys, ε-Lys, ζ-D-Orn, ζ-Orn, γ-aminobutyric acid,8-aminooctanoic acid, 11-amino-undecanoic acid,8-amino-3,6-dioxaoctanoic acid, 4-amino-4-carboxylic piperidine andbis(D-Lys-Gly)Lactam.

Or (ii) G is

and p is 1; The linking moiety, W can be any of the following threealternatives: (a) null, provided that when W is null, Y is nitrogen andis bonded to the C-terminus of Xaa₄ to form an amide; (b) —NH—(CH₂)_(b)—with b equal to 0, 1, 2, 3, 4, 5, or 6; or (c) —NH—(CH₂)_(c)—O— with cequal to 2, or 3, provided that Y is carbon. In each of the foregoingalternatives, (b) and (c) the nitrogen atom of W is bonded to theC-terminus of Xaa₄ to form an amide; and the moiety

is an optionally substituted 4-, 5-, 6-, 7-, or 8-membered heterocyclicring moiety wherein Y is a carbon or a nitrogen atom and Z is carbon,nitrogen, oxygen, sulfur, sulfoxide, or sulfonyl; provided that whensuch ring moiety is a six, seven or eight-membered ring, Y and Z areseparated by at least two ring atoms, and provided further that whensuch ring moiety is aromatic, then Y is a carbon atom.

The moiety V in the substituent of the Y-Z-containing ring in formula Iis a C₁-C₆ alkyl linker when present. The operator, e is zero or 1, suchthat when e is zero, then V is null, and R₁ and R₂ are directly bondedto the same or different ring atoms. The moiety V represents C₁-C₆alkyl, and the operator, e is either zero or 1, wherein when e is zero,then V is null and, R₁ and R₂ are directly bonded to the same ordifferent ring atoms. The groups R₁ and R₂ can be any one of (a), (b),(c) or (d) as follows:

(a) R₁ is H, OH, halo, CF₃, —NH₂, —COOH, C₁-C₆ alkyl, C₁-C₆ alkoxy,amidino, C₁-C₆ alkyl-substituted amidino, aryl, optionally substitutedheterocyclyl, Pro-amide, Pro, Gly, Ala, Val, Leu, Ile, Lys, Arg, Orn,Ser, Thr, CN, CONH₂, COR′, SO₂R′, CONR′R″, NHCOR′, OR′, or SO₂NR′R″;wherein said optionally substituted heterocyclyl is optionally singly ordoubly substituted with substituents independently selected from thegroup consisting of C₁-C₆ alkyl, —C₁-C₆ alkoxy, oxo, —OH, —Cl, —F, —NH₂,—NO₂, —CN, —COOH, and amidino; wherein R′ and R″ are each independentlyH, C₁-C₈ alkyl, aryl, heterocyclyl or R′ and R″ are combined to form a4-, 5-, 6-, 7-, or 8-membered ring, which ring is optionally substitutedsingly or doubly with substituents independently selected from the groupconsisting of C₁-C₆ alkyl, —C₁-C₆ alkoxy, —OH, —Cl, —F, —NH₂, —NO₂, —CN,and —COOH, amidino; and R₂ is H, amidino, singly or doubly C₁-C₆alkyl-substituted amidino, —CN, —CONH₂, —CONR′R″, —NHCOR′, —SO₂NR′R″, or—COOH; or

(b) R₁ and R₂ taken together can form an optionally substituted 4-, 5-,6-, 7-, 8- or 9-membered heterocyclic monocyclic or bicyclic ring moietywhich is bonded to a single ring atom of the Y and Z-containing ringmoiety; or

(c) R₁ and R₂ taken together with a single ring atom of the Y andZ-containing ring moiety can form an optionally substituted 4-, 5-, 6-,7-, or 8-membered heterocyclic ring moiety to form a spiro structure; or

(d) R₁ and R₂ taken together with two or more adjacent ring atoms of theY and Z-containing ring moiety can form an optionally substituted 4-,5-, 6-, 7-, 8- or 9-membered hetero-cyclic monocyclic or bicyclic ringmoiety fused to the Y and Z-containing ring moiety.

Each of the aforementioned optionally substituted 4-, 5-, 6-, 7-, 8- or9-membered heterocyclic ring moieties that include R₁ and R₂ isoptionally singly or doubly substituted with substituents independentlychosen from C₁-C₆ alkyl, C₁-C₆ alkoxy, optionally substituted phenyl (asdefined above), oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH, and amidino.

In the first of four alternative embodiments, the moiety R₁ in formula Ican be any of the following groups: —H, —OH, halo, —CF₃, —NH₂, —COOH,C₁-C₆ alkyl, C₁-C₆ alkoxy, amidino, C₁-C₆ alkyl-substituted amidino,aryl, optionally substituted heterocyclyl, Pro-amide, Pro, Gly, Ala,Val, Leu, Ile, Lys, Arg, Orn, Ser, Thr, CN, CONH₂, COR′, SO₂R′, CONR′R″,NHCOR′, OR′, or SO₂NR′R″; wherein the optionally substitutedheterocyclyl is optionally singly or doubly substituted withsubstituents independently chosen from C₁-C₆ alkyl, C₁-C₆ alkoxy, oxo,—OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH, and amidino. The moieties R′ andR″ are each independently H, C₁-C₈ alkyl, aryl, or heterocyclyl.Alternatively, R′ and R″ can be combined to form a 4-, 5-, 6-, 7-, or8-membered ring, which ring is optionally substituted singly or doublywith substituents independently chosen from C₁-C₆ alkyl, C₁-C₆ alkoxy,—OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH and amidino. The moiety R₂ can beany of —H, amidino, singly or doubly C₁-C₆ alkyl-substituted amidino,—CN, —CONH₂, —CONR′R″, —NHCOR′, —SO₂NR′R″, or —COOH.

In a second alternative embodiment, the moieties R₁ and R₂ takentogether can form an optionally substituted 4-, 5-, 6-, 7-, 8- or9-membered heterocyclic monocyclic or bicyclic ring moiety which isbonded to a single ring atom of the Y and Z-containing ring moiety.

In a third alternative embodiment, the moieties R₁ and R₂ taken togetherwith a single ring atom of the Y and Z-containing ring moiety can forman optionally substituted 4-, 5-, 6-, 7- or 8-membered heterocyclic ringmoiety to form a spiro structure.

In a fourth alternative embodiment, the moieties R₁ and R₂ takentogether with two or more adjacent ring atoms of the Y and Z-containingring moiety can form an optionally substituted 4-, 5-, 6-, 7-, 8- or9-membered heterocyclic monocyclic or bicyclic ring moiety fused to theY and Z-containing ring moiety.

In formula I in the above second, third and fourth alternativeembodiments, each of the optionally substituted 4-, 5-, 6-, 7-, 8- and9-membered heterocyclic ring moieties comprising R₁ and R₂ can be singlyor doubly substituted with substituents independently chosen from C₁-C₆alkyl, C₁-C₆ alkoxy, optionally substituted phenyl, oxo, —OH, —Cl, —F,—NH₂, —NO₂, —CN, —COOH and amidino.

Alternatively, (iii) G is

wherein J is a 5-, 6-, or 7-membered heterocyclic ring moiety comprising1, 2, or 3 heteroatoms in the ring wherein R₃ and R₄ are eachindependently selected from H, C₁-C₃ alkyl, halo, —OH, —CF₃, —NH₂, —COOHand amidino; and R₅ and R₆ are each independently selected from H, C₁-C₃alkyl, oxo, halo, —OH, —CF₃, —NH₂, —COOH and amidino.

The moiety W′ is chosen from the following two options: —NH—(CH₂)_(b)—with b equal to zero, 1, 2, 3, 4, 5, or 6; and —NH—(CH₂)_(c)—O— with cequal to 2 or 3.

The above formula I definitions are subject to the following threeprovisos:

(1) That when the Y and Z-containing ring moiety is a six or sevenmembered ring having a single ring heteroatom and such heteroatom is N,and e is zero, then R₁ is not OH, and R₁ and R₂ are not both H;

(2) That when the Y and Z-containing ring moiety is a six membered ringcomprising two ring heteroatoms, both Y and Z are nitrogen atoms, W isnull, and the moiety —V_(e)(R₁)(R₂) is attached to Z, then—V_(e)(R₁)(R₂) is chosen from amidino, C₁-C₆ alkyl-substituted amidino,dihydroimidazole, —CH₂COOH, and —CH₂C(O)NH₂; and

(3) That if the Y and Z-containing ring moiety is a six membered ringcomprising a sulfur or an oxygen ring heteroatom, or if the Y andZ-containing ring moiety is a non-aromatic six membered ring thatincludes two ring heteroatoms, wherein both Y and Z are nitrogen atomsand W is null, or if the Y and Z-containing ring moiety is asix-membered aromatic ring that includes a single ring heteroatom, whichheteroatom is a nitrogen atom, then, when e is zero, R₁ and R₂ are notboth hydrogen.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Concentration detected in rat plasma and brain afteradministration of 3 mg/kg compound (2) over a 5 minute infusion througha jugular vein catheter. Concentration of compound (2) in ng/ml: opencircles: plasma, solid circles: brain.

FIG. 2: Plasma concentrations of compound (6) after subcutaneousadministration of a single bolus of 1 mg/kg of the compound to ICR mice.Plasma was sampled at 5, 10, 15, 20, 30 60, 90 120, and 180 minutespost-injection.

FIG. 3: Plasma concentrations of compound (3) after intravenousadministration of a single bolus of 0.56 mg/kg of the compound tocynomolgus monkeys. Plasma was sampled at 2, 5, 10, 15, 30, 60, 120, and240 minutes post injection.

FIG. 4: Dose-response curves for compound (3) in ICR mice in the aceticacid-induced writhing assay (solid circles) and in the locomotion assay(solid squares).

FIG. 5: Dose response of compound (2)-mediated suppression of aceticacid-induced writhing in mice when delivered by the intravenous route.

FIG. 6: Dose response curves for compound (54) in IRC mice in the aceticacid-induced writhing assay (open circles) and the mean (closed circles)and error bars; and in the locomotion assay (open squares) and the mean(closed squares) and error bars.

FIG. 7: Plasma concentrations after intravenous administration of asingle bolus of compound (52) to cynomolgus monkeys. Plasma was sampledat 2, 5, 10, 15, 30, 60, 120, and 240 minutes post injection.

FIG. 8: Effects of compound (2) on mechanical hypersensitivity inducedby L5/L6 spinal nerve ligation in rats. Open circles—vehicle alone;Solid circles—compound (2) at 0.1 mg/kg; open squares—compound (2) at0.3 mg/kg; solid squares—compound (2) at 1.0 mg/kg. ** denotes p<0.01;*** denotes p<0.001 vs. Vehicle (2-way ANOVA, Bonferroni).

FIG. 9: Effect of compound (2) on pancreatitis-induced abdominalhypersensitivity in rats. Dibutyltin dichloride or vehicle alone wasadministered intravenously and hypersensitivity assessed by abdominalprobing with a von Frey filament at 30 minute intervals.Hypersensitivity is expressed as number of withdrawals from tenprobings. Open circles—vehicle alone; solid circles—compound (2) at 0.1mg/kg; open squares—compound (2) at 0.3 mg/kg; solid squares—compound(2) at 1.0 mg/kg. ** denotes p<0.01; *** denotes p<0.001 vs. Vehicle(2-way ANOVA, Bonferroni).

FIG. 10: Blocking of the effect of compound (2) on pancreatitis-inducedabdominal hypersensitivity by nor-BNI and naloxone methiodide (NM) inrats. Open column—vehicle alone, solid column—compound (2) at 1 mg/kgwith NM or norBNI as indicated. *** denotes p<0.001 vs. Vehicle+Vehicle(2-way ANOVA, Bonferroni).

FIG. 11: Inhibition of proliferation of human synoviocytes afterincubation with Compound (2) in vitro.

DETAILED DESCRIPTION

As used throughout this specification, the term “synthetic peptideamide” means a compound of the invention conforming to formula I, or astereoisomer, mixture of stereoisomers, prodrug, pharmaceuticallyacceptable salt, hydrate, solvate, acid salt hydrate, N-oxide orisomorphic crystalline form thereof. The designations Xaa₁, Xaa₂, Xaa₃,and Xaa₄ represent D-amino acids in the synthetic peptide amides of theinvention. Stereoisomers of the synthetic peptide amides of theinvention conforming to formula I are limited to those compounds havingamino acids in the D-configuration where so specified in Formula I.Stereoisomers of the synthetic peptide amides of the invention includecompounds having either a D- or L-configuration at chiral centers otherthan the alpha carbons of the four amino acids at Xaa₁, Xaa₂, Xaa₃, andXaa₄. The term ‘mixtures of stereoisomers’ refer to mixtures of suchstereoisomers of the invention. As used herein ‘racemates’ refers tomixtures of stereoisomers having equal proportions of compounds with D-and L-configuration at one or more of the chiral centers other than thealpha carbons of Xaa₁, Xaa₂, Xaa₃, and Xaa₄ without varying thechirality of the alpha carbons of Xaa₁, Xaa₂, Xaa₃, and Xaa₄.

The nomenclature used to define peptides herein is specified by Schroder& Lubke, The Peptides, Academic Press, 1965, wherein, in accordance withconventional representation, the N-terminus appears to the left and theC-terminus to the right. Where an amino acid residue has isomeric forms,both the L-isomer form and the D-isomer form of the amino acid areintended to be covered unless otherwise indicated Amino acids arecommonly identified herein by the standard three-letter code. TheD-isomer of an amino acid is specified by the prefix “D-” as in “D-Phe”which represents D-phenylalanine, the D-isomer of phenylalanine.Similarly, the L-isomer is specified by the prefix “L-” as in “L-Phe.”Peptides are represented herein according to the usual convention asamino acid sequences from left to right: N-terminus to C-terminus,unless otherwise specified.

As used herein, D-Arg represents D-arginine, D-Har representsD-homoarginine, which has a side chain one methylene group longer thanD-Arg, and D-Nar represents D-norarginine, which has a side chain onemethylene group shorter than D-Arg. Similarly, D-Leu means D-leucine,D-Nle means D-norleucine, and D-Hle represents D-homoleucine. D-Alameans D-alanine, D-Tyr means D-tyrosine, D-Trp means D-tryptophan, andD-Tic means D-1,2,3,4-tetrahydroisoquinoline-3carboxylic acid. D-Valmeans D-valine and D-Met means D-methionine. D-Pro means D-proline,Pro-amide means the D- or L-form of proline amide. D-Pro amiderepresents D-proline with an amide formed at its carboxy moiety whereinthe amide nitrogen may be alkyl substituted, as in —NR_(a)R_(b), whereinR_(a) and R_(b) are each independently a C₁-C₆ alkyl group, or one ofR_(a) and R_(b) is —H. Gly means glycine, D-Ile means D-isoleucine,D-Ser means D-serine, and D-Thr means D-threonine. (E)D-Ala means theD-isomer of alanine which is substituted by the substituent (E) on theβ-carbon. Examples of such substituent (E) groups include tert-butyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, furyl, pyridyl,thienyl, thiazolyl and benzothienyl. Thus, cyclopentyl-D-Ala means theD-isomer of alanine which is substituted by cyclopentyl on the β-carbon.Similarly, D-Ala(2-thienyl) and (2-thienyl)D-Ala are interchangeable andboth mean the D-isomer of alanine substituted at the β-carbon withthienyl that is attached at the 2-ring position.

As used herein, D-Nal means the D-isomer of alanine substituted bynaphthyl on the β-carbon. D-2Nal means naphthyl substituted D-alaninewherein the attachment to naphthalene is at the 2-position on the ringstructure and D-1Nal means naphthyl-substituted D-alanine wherein theattachment to naphthalene is at the 1-position on the ring structure. By(A)(A′)D-Phe is meant D-phenylalanine substituted on the phenyl ringwith one or two substituents independently chosen from halo, nitro,methyl, halomethyl (such as, for example, trifluoromethyl),perhalomethyl, cyano and carboxamide. By D-(4-F)Phe is meantD-phenylalanine which is fluoro-substituted in the 4-position of thephenyl ring. By D-(2-F)Phe is meant D-phenylalanine which isfluoro-substituted in the 2-position of the phenyl ring. By D-(4-Cl)Pheis meant D-phenylalanine which is chloro substituted in the 4-phenylring position. By (α-Me)D-Phe is meant D-phenylalanine which is methylsubstituted at the alpha carbon. By (α-Me)D-Leu is meant D-leucine whichis methyl substituted at the alpha carbon.

The designations (B)₂D-Arg, (B)₂D-Nar, and (B)₂D-Har representD-arginine, D-norarginine and D-homoarginine, respectively, each havingtwo substituent (B) groups on the side chain. D-Lys means D-lysine andD-Hlys means D-homolysine. ζ-(B)D-Hlys, ε-(B)D-Lys, and ε-(B)₂-D-Lysrepresent D-homolysine and D-lysine each having the side chain aminogroup substituted with one or two substituent (B) groups, as indicated.D-Orn means D-ornithine and δ-(B)α-(B′)D-Orn means D-ornithinesubstituted with (B′) at the alpha carbon and substituted with (B) atthe side chain ζ-amino group.

D-Dap means D-2,3-diaminopropionic acid. D-Dbu represents the D-isomerof alpha, gamma-diamino butyric acid and (B)₂D-Dbu represents alpha,gamma-diamino butyric acid which is substituted with two substituent (B)groups at the gamma amino group. Unless otherwise stated, each of the(B) groups of such doubly substituted residues are independently chosenfrom H— and C₁-C₄-alkyl. As used herein, D-Amf means D-(NH₂CH₂-)Phe,i.e., the D-isomer of phenylalanine substituted with aminomethyl on itsphenyl ring and D-4Amf represents the particular D-Amf in which theaminomethyl is attached at the 4-position of the ring. D-Gmf meansD-Amf(amidino) which represents D-Phe wherein the phenyl ring issubstituted with —CH₂NHC(NH)NH₂. Amd represents amidino, —C(NH)NH₂, andthe designations (Amd)D-Amf and D-Amf(Amd) are also interchangeably usedfor D-Gmf. The designations Ily and Ior are respectively used to meanisopropyl Lys and isopropyl Orn, wherein the side chain amino group isalkylated with an isopropyl group.

Alkyl means an alkane radical which can be a straight, branched, andcyclic alkyl group such as, but not limited to, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, t-butyl, sec-butyl, pentyl, cyclopentyl,hexyl, cyclohexyl, cyclohexylethyl. C₁ to C₈ alkyl refers to alkylgroups having between one and eight carbon atoms. Similarly, C₁-C₆ alkylrefers to alkyl groups having between one and six carbon atoms.Likewise, C₁-C₄ alkyl refers to alkyl groups having between one and fourcarbon atoms. By lower alkyl is meant C₁-C₆ alkyl. Me, Et, Pr, Ipr, Bu,and Pn are interchangeably used to represent the common alkyl groups:methyl, ethyl, propyl, isopropyl, butyl, and pentyl, respectively.Although the linkage for an alkyl group is typically at one end of analkyl chain, the linkage may be elsewhere in the chain, e.g. 3-pentylwhich may also be referred to as ethylpropyl, or 1-ethylprop-1-yl.Alkyl-substituted, such as C₁ to C₆ alkyl-substituted amidino, indicatesthat the relevant moiety is substituted with one or more alkyl groups.

Where a specified moiety is null, the moiety is absent and if suchmoiety is indicated to be attached to two other moieties, such two othermoieties are connected by one covalent bond. Where a connecting moietyis shown herein as attached to a ring at any position on the ring, andattached to two other moieties, such as R₁ and R₂, in the case where theconnecting moiety is specified to be null, then the R₁ and R₂ moietiescan each be independently attached to any position on the ring.

The terms “heterocycle”, “heterocyclic ring” and “heterocyclyl” are usedinterchangeably herein and refer to a ring or ring moiety having atleast one non-carbon ring atom, also called a heteroatom, which can be anitrogen atom, a sulfur atom, or an oxygen atom. Where a ring isspecified as having a certain number of members, the number defines thenumber of ring atoms without reference to any substituents or hydrogenatoms bonded to the ring atoms. Heterocycles, heterocyclic rings andheterocyclyl moieties can include multiple heteroatoms independentlyselected from nitrogen, sulfur, or oxygen atom in the ring. Rings can besubstituted at any available position. For example, but withoutlimitation, 6- and 7-membered rings are often substituted in the 4-ringposition and 5-membered rings are commonly substituted in the3-position, wherein the ring is attached to the peptide amide chain atthe 1-ring position.

The term “saturated” means an absence of double or triple bonds and theuse of the term in connection with rings describes rings having nodouble or triple bonds within the ring, but does not preclude double ortriple bonds from being present in substituents attached to the ring.The term “non-aromatic” in the context of a particular ring refers to anabsence of aromaticity in that ring, but does not preclude the presenceof double bonds within the ring, including double bonds which are partof an aromatic ring fused to the ring in question. Nor is a ring atom ofa saturated heterocyclic ring moiety precluded from being double-bondedto a non-ring atom, such as for instance a ring sulfur atom beingdouble-bonded to an oxygen atom substituent. As used herein,heterocycles, heterocyclic rings and heterocyclyl moieties also includesaturated, partially unsaturated and heteroaromatic rings and fusedbicyclic ring structures unless otherwise specified. A heterocycle,heterocyclic ring or heterocyclyl moiety can be fused to a second ring,which can be a saturated, partially unsaturated, or aromatic ring, whichring can be a heterocycle or a carbocycle. Where indicated, twosubstituents can be optionally taken together to form an additionalring. Rings may be substituted at any available position. A heterocycle,heterocyclic ring and heterocyclyl moiety can, where indicted, beoptionally substituted at one or more ring positions with one or moreindependently selected substituents, such as for instance, C₁-C₆ alkyl,C₃-C₈ cycloalkyl, C₁-C₆ alkoxy, halo C₁-C₆ alkyl, optionally substitutedphenyl, aryl, heterocyclyl, oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOHand amidino. Suitable optional substituents of the phenyl substituentinclude for instance, but without limitation, one or more groupsselected from C₁-C₃ alkyl, C₁-C₃ alkoxy, halo C₁-C₃ alkyl, oxo, —OH,—Cl, —F, —NH₂, —NO₂, —CN, —COOH and amidino.

D-Phe and substituted D-Phe are examples of a suitable amino acid forresidue Xaa₁ in Formula I. The phenyl ring can be substituted at any ofthe 2-, 3- and/or 4-positions. Particular examples of permittedsubstitutions include, for instance, chlorine or fluorine at the 2- or4-positions. Also the alpha-carbon atom may be methylated. Otherequivalent residues which represent conservative changes to D-Phe canalso be used. These include D-Ala(cyclopentyl), D-Ala(thienyl), D-Tyrand D-Tic. The residue at the second position, Xaa₂ can also be D-Phe orsubstituted D-Phe with such substitutions including a substituent on the4-position carbon of the phenyl ring, or on both the 3- and 4-positions.Alternatively, Xaa₂ can be D-Trp, D-Tyr or D-alanine substituted bynaphthyl. The third position residue, Xaa₃ can be any non-polar aminoacid residue, such as for instance, D-Nle, D-Leu, (α-Me)D-Leu, D-Hle,D-Met or D-Val. However, D-Ala (cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl) or D-Phe can also be used as Xaa₃. The fourth positionresidue Xaa₄ can be any positively charged amino acid residue, such asfor instance, D-Arg and D-Har, which can be optionally substituted withlower alkyl groups, such as one or two ethyl groups. Alternatively,D-Nar and any other equivalent residues can be used, such as, forinstance, D-Lys or D-Orn (either of which can be w-amino groupalkylated, for example by methyl or isopropyl groups, or methylated atthe α-carbon group). Moreover, D-Dbu, D-4-Amf (which can be optionallysubstituted with amidino), and D-Hlys are also suitable amino acids atthis position.

Compounds of the invention contain one or more chiral centers, each ofwhich has two possible three-dimensional spatial arrangements(configurations) of the four substituents around the central carbonatom. These are known as “stereoisomers”, and more specifically as“enantiomers” (all chiral centers inverted) or “diastereoisomers” (twoor more chiral centers, at least one chiral center remaining the same).In a specific embodiment of the invention, the amino acids which make upthe tetrapeptide backbone, Xaa₁Xaa₂Xaa₃Xaa₄ are specified to be D-aminoacids i.e., the opposite configuration to those generally found inmammals. Reference to stereoisomers of the synthetic peptide amides ofthe invention concerns chiral centers other than the alpha carbons ofthe D-amino acids which make up Xaa₁-Xaa₄. Thus, stereoisomers ofsynthetic peptide amides that are embodiments of the invention whereineach of Xaa₁-Xaa₄ are specified to be D-amino acids, do not includeL-amino acids or racemic mixtures of the amino acids at these positions.Similarly, reference to racemates herein concerns a center other thanthe alpha carbons of the D-amino acids which make up Xaa₁-Xaa₄. Chiralcenters in the synthetic peptide amides of the invention for which astereoisomer may take either the R or S configuration include chiralcenters in the moiety attached to the carboxy-terminus of Xaa₄, and alsochiral centers in any amino acid side chain substituents of Xaa₁-Xaa₄.

The synthetic peptide amides of the invention described herein (alsointerchangeably referred to as synthetic peptide amide compounds,compounds of the invention, compound (number), or simply “thecompounds”) can be used or prepared in alternate forms. For example,many amino-containing compounds can be used or prepared as an acid salt.Often such salts improve isolation and handling properties of thecompound. For example, depending on the reagents, reaction conditionsand the like, compounds such as the synthetic peptide amides describedherein can be used or prepared, for example, as the hydrochloride ortosylate salts. Isomorphic crystalline forms, all chiral and racemicforms, N-oxide, hydrates, solvates, and acid salt hydrates, are alsocontemplated to be within the scope of the present invention.

Certain acidic or basic synthetic peptide amides of the presentinvention may exist as zwitterions. All forms of these synthetic peptideamide compounds, including free acid, free base and zwitterions, arecontemplated to be within the scope of the present invention. It is wellknown in the art that compounds containing both amino and carboxylgroups often exist in equilibrium with their zwitterionic forms. Thus,for any compound described herein that contains, for example, both aminoand carboxyl groups, it will also be understood to include thecorresponding zwitterion.

As used herein, the chemical designation“tetrapeptide-[ω(4-amino-piperidine-4-carboxylic acid)]” is used toindicate the aminoacyl moiety of the synthetic peptide amides of theinvention derived from 4-aminopiperidine-4-carboxylic acid, wherein thenitrogen atom of the piperidine ring is bound to the C-terminalcarbonyl-carbon of the tetrapeptide fragment, unless otherwiseindicated.

In one embodiment, the invention provides a synthetic peptide amidewherein each Xaa₁ is D-Phe, each Xaa₂ is D-Phe, each Xaa₃ is D-Leu andeach Xaa₄ is D-Lys. In another embodiment, each Xaa₁ isD-Ala(2-thienyl), each Xaa₂ is D-Phe, each Xaa₃ is D-Nle, and each Xaa₄is D-Arg.

In another embodiment, G is

and the dipeptide Xaa₃-Xaa₄ is chosen from D-Leu-D-Orn and D-Nle-D-Arg.In another embodiment Xaa₁₋Xaa₂ is D-Phe-D-Phe. In another embodimentXaa₁ is D-(4-F)Phe, and Xaa₂ is D-(4-Cl)Phe.

In another embodiment each Xaa₁ is D-Phe or D-Ala(2-thienyl) and eachXaa₂ is D-(4-Cl)Phe. In another embodiment, each Xaa₃ is D-Leu or D-Nle.

In another embodiment G is

and Xaa₁ is chosen from D-Phe, D-(4-F)Phe, D-(2-F)Phe, cyclopentylD-Ala, 2-thienyl D-Ala, Xaa₂ is chosen from D-Phe, D-(4-F)Phe,D-(4-Cl)Phe, D-1Nal, D-2Nal, and D-Trp, and Xaa₃-Xaa₄ is chosen fromD-Nle-D-Arg, D-Leu-D-Lys and D-Leu-D-Orn.

In one embodiment Xaa₁ is (A)(A′)D-Phe, and in one aspect, each Xaa₁ isD-Phe. In another embodiment each Xaa₂ is D-Phe. In another embodiment,each Xaa₃ is chosen from D-Nle, and D-Leu. In another embodiment eachXaa₄ is chosen from δ(B)₂D-Orn, D-Lys and D-Arg. In one aspect, eachXaa₄ is δ(B)₂D-Orn and each (B) chosen from —H, methyl and isopropyl. Inanother aspect each Xaa₄ is (B)₂D-Orn, wherein one (B) is H, and theother (B) selected from the group consisting of methyl and isopropyl. Inanother particular aspect each Xaa₄ is D-Orn.

In another embodiment each Xaa₄ is chosen from ε(B)2D-Lys, (B)₂D-Arg,and δ-(B)₂D-Orn. In another particular aspect each Xaa₄ is chosen fromD-Arg, (Et)₂D-Arg, and δ-(B)D-Orn, and (B) is H, Me, iPr, or Bu.

In another embodiment G is

and W is null.

In another embodiment G is

and W is —N—(CH₂)_(b) with b equal to 0, 1, 2, 3, or 4. In one aspect bis zero and Y is a carbon atom. In another aspect b is 1 or 2 and Y is anitrogen atom. In another embodiment W is —N—(CH₂)_(c)—O—. In oneparticular aspect c is 1 or 2. In another aspect the Y and Z-containingring moiety is a four or five membered ring and Y is a nitrogen atom. Inanother embodiment the Y and Z-containing ring moiety is a four or fivemembered ring and Y is a carbon atom.

In another embodiment the Y and Z-containing ring moiety is a six orseven membered ring, Y is nitrogen and Z is a carbon atom. In anotheralternative, the Y and Z-containing ring moiety is a six membered ring.In one aspect the Y and Z-containing ring moiety is a seven memberedring. In still another aspect the Y and Z-containing ring moiety is asix or seven membered ring and both Y and Z are nitrogen atoms.

In another embodiment e is zero and R₁ and R₂ are bonded directly to thesame ring atom. In one aspect e is zero, R₂ is —H and R₁ is bondeddirectly to a carbon ring atom adjacent to Z. In another aspect R₁ is H,amidino, C₁-C₃ alkyl substituted amidino, C₁-C₃ alkyl, dihydroimidazole,D-Pro, D-Pro amide, or —CONH₂ and wherein e is zero and R₂ is —H. Inanother aspect R₁ is —H, amidino, or methyl amidino. In one aspect the Yand Z-containing ring moiety is a five membered ring, e is zero and R₁is —COOH.

In another embodiment G is

and Xaa₁ is D-Phe, Xaa₂ is D-Phe, Xaa₃ is D-Leu, Xaa₄ is ε(B)2D-Lys, orδ-(B)₂D-Orn, wherein (B) is —H, methyl, or isopropyl; further wherein Wis null, the Y and Z-containing ring moiety is a six or seven memberedring, Y is a nitrogen atom, e is zero, R₁ is —NH₂, amidino, C₁-C₃ alkyl,C₁-C₃ alkyl-substituted amidino, dihydroimidazole, D-Pro, or D-Proamide, and R₂ is H or —COOH.

In another embodiment, G is chosen from the following groups:

In certain embodiments of the synthetic peptide amides of the invention,there are two independent occurrences of the residues Xaa₁, Xaa₂, Xaa₃and Xaa₄. For instance, in embodiments having the formula:

wherein G is:

and one or more of q, r, and s is 1 or both p and t are 1, then thereare two occurrences of Xaa₁, Xaa₂, Xaa₃ and Xaa₄ respectively. In suchembodiments, each instance of each of the residues Xaa₁, Xaa₂, Xaa₃ andXaa₄ can be identical.

Alternatively, and in other embodiments, each instance of one or more ofthe pairs of residues Xaa₁, Xaa₂, Xaa₃ or Xaa₄ can be different. Forexample, one instance of Xaa₁ can be D-phenylalanine, while the secondinstance of Xaa₁ in the same molecule can be a different Xaa₁ residue,such as D-(4-F)phenylalanine. Similarly, one instance of Xaa₂ can beD-phenylalanine, while the second instance of Xaa₂ in the same moleculecan be D-Ala(2-thienyl). Likewise, one instance of Xaa₃ can beD-norleucine, while the second instance of Xaa₃ in the same molecule canbe D-leucine. In the same manner, one instance of Xaa₄ can beD-ornithine, while the second instance of Xaa₄ in the same molecule canbe D-arginine, and so on.

In one embodiment, the invention provides a synthetic peptide amidewherein Xaa₁ is D-Ala(2-thienyl). In another embodiment Xaa₁ isD-(4-F)phenylalanine and Xaa₂ is D-(4-Cl)phenylalanine. In anotherembodiment each Xaa₁ is D-phenylalanine or D-Ala(2-thienyl) and eachXaa₂ is D-(4-Cl)phenylalanine. In another embodiment Xaa₁₋Xaa₂ isD-phenylalanine-D-phenylalanine.

In one embodiment each Xaa₃ is chosen from D-norleucine and D-leucine.In another embodiment each Xaa₂ is D-phenylalanine, each Xaa₃ isD-norleucine, and each Xaa₄ is D-arginine. In another embodiment eachXaa₃ can be D-leucine or D-norleucine.

In another embodiment Xaa₄ is chosen from δ(B)₂D-ornithine andD-arginine. Alternatively, each Xaa₄ is δ(B)₂D-ornithine and each (B) ischosen from —H, methyl and isopropyl. In still another embodiment, eachXaa₄ is (B)₂D-ornithine, wherein one (B) is —H, and the other (B) chosenfrom methyl and isopropyl. In one aspect, each Xaa₄ is (B)₂D-arginine,or δ-(B)₂D-ornithine. In another embodiment each Xaa₄ can be a residuechosen from D-arginine, (Et)₂D-arginine, and δ-(B)D-ornithine, andwherein (B) is —H, methyl, isopropyl, or butyl. In one embodiment thedipeptide Xaa₃-Xaa₄ is chosen from D-leucine-D-ornithine andD-norleucine-D-arginine.

In one particular embodiment the synthetic peptide amide of theinvention has the formula

wherein G is:

and b is zero and Y is a carbon atom. In another embodiment, b is 1 or 2and Y is a nitrogen atom. In a particular aspect of the invention, b is2.

In another embodiment G is

and the Y- and Z-containing moiety is [ω(4-aminopiperidine-4-carboxylicacid)]-OH.

In one particular embodiment Xaa₁ is chosen from D-Phe, D-(4-F)Phe,D-(2-F)Phe, cyclopentyl D-Ala, 2-thienyl D-Ala, Xaa₂ is chosen fromD-(4-F)Phe, D-(4-Cl)Phe, D-1Nal, D-2Nal, and D-Trp, and Xaa₃-Xaa₄ ischosen from D-Nle-D-Arg and D-Leu-D-Orn.

In another embodiment W is an N-alkoxyl linker of the formula:—N—(CH₂)₂—O—. In an alternative embodiment W is null andXaa₁Xaa₂Xaa₃Xaa₄ is directly bonded to Y. In a second alternativeembodiment, W is —NH—(CH₂)₂—.

In another particular embodiment, the Y and Z-containing ring moiety isa four or five membered ring and Y is a nitrogen atom. Alternatively,the Y- and Z-containing ring moiety can be a four or five membered ringwherein Y is a carbon atom. In a different embodiment, the Y andZ-containing ring moiety is a 6- or 7-membered ring, Y is a nitrogenatom and Z is a carbon atom. In one aspect of this embodiment, the Y andZ-containing ring moiety is a 6-membered ring. Alternatively, the Y andZ-containing ring moiety can be a seven membered ring. In one aspect ofthis embodiment, the Y and Z-containing ring moiety is a 6- or7-membered ring and both Y and Z are nitrogen atoms.

In another particular embodiment the Y- and Z-containing ring moiety isa six or seven membered ring, or an eight-membered ring, Y is a carbonatom, and Z is a nitrogen atom. In one aspect, Y is a nitrogen atom andZ is a carbon atom. In an alternative embodiment Y and Z are eachnitrogen atoms.

In another particular embodiment the Y- and Z-containing ring moiety isan optionally substituted 4-, 5-, 6-, 7-, or 8-membered heterocyclicring moiety wherein Y is a carbon or a nitrogen atom and Z is carbon,nitrogen, oxygen, sulfur, sulfoxide, or sulfonyl; and the 4-, 5-, 6-,7-, or 8-membered heterocyclic ring moiety is optionally singly ordoubly substituted with substituents independently chosen from C₁-C₆alkyl, —C₁-C₆ alkoxy, oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH, andamidino. In one aspect when the Y- and Z-containing ring moiety is asix, seven or eight-membered ring, then Y and Z are separated by atleast two ring atoms. In another aspect, when the Y- and Z-containingring moiety is non-aromatic and Z is a carbon or a nitrogen atom, thensuch ring moiety includes at least one sulfur or oxygen ring heteroatom.In a particular aspect, when the Y- and Z-containing ring moiety isaromatic, then Y is a carbon atom.

In one embodiment of the synthetic peptide amide of the invention, R₁ is—H, —OH, —NH₂, —COOH, C₁-C₃ alkyl, amidino, C₁-C₃ alkyl-substitutedamidino, dihydroimidazole, D-Pro, D-Pro amide, or —CONH₂. In anotherparticular embodiment R₂ is —H, —COOH, or C₁-C₃ alkyl. In one aspect,only one of R₁ and R₂ is a hydrogen atom. In a particular embodiment R₁is —H, D-Pro, D-Pro amide, or —NH₂ and R₂ is H or —COOH. In one aspectof this embodiment, R₁ is —NH₂ and ₂ is —COOH.

In one embodiment, the operator, e is zero and R₁ and R₂ are bondeddirectly to the same ring atom. In a particular embodiment, e is zero,R₂ is —H and R₁ is bonded directly to a carbon ring atom adjacent to Z.In another particular embodiment, R₁ is —H, amidino, C₁-C₃ alkylsubstituted amidino, C₁-C₃ alkyl, dihydroimidazole, D-Pro, D-Pro amide,or —CONH₂ and e is zero and R₂ is —H.

In one embodiment of the synthetic peptide amide of the invention, Xaa₁is D-Phe, Xaa₂ is D-Phe, Xaa₃ is D-Leu, Xaa₄ is δ-(B)₂D-Orn, wherein (B)is —H, methyl, or isopropyl; such that wherein W is null, the Y andZ-containing ring moiety is a six or seven membered ring, Y is an Natom, e is zero, R₁ is NH₂, amidino, C₁-C₃ alkyl, C₁-C₃alkyl-substituted amidino, dihydroimidazole, D-Pro, or D-Pro amide, andR₂ is H or COOH.

In one embodiment of the synthetic peptide amide of the invention: Xaa₁is chosen from (A) D-Phe, (α-Me)D-Phe, D-Tyr, D-Tic, (tert-butyl)D-Gly,and β-(E)D-Ala, wherein (A) is chosen from —H, —F, —Cl, —NO₂, and —CH₃,and (E) is chosen from tert-butyl, cyclopentyl and thienyl; Xaa₂ ischosen from (A)(A′)D-Phe, D-1Nal, D-2Nal, D-Tyr, and D-Trp, wherein (A′)is H or Cl; Xaa₃ is chosen from D-Nle, D-Phe, (cyclopentyl) D-Ala,D-Leu, (α-Me)D-Leu, D-Hle, D-Val, and D-Met; and Xaa₄ is chosen fromD-Arg, (ethyl)₂₋D-Arg, D-Nar, D-Har, (ethyl)₂D-Har, ε-(isopropyl)D-Lys,D-Lys, D-Amf, amidino-D-Amf, β-amidino-D-Dap, D-Dbu, D-Orn,α-(methyl)D-Orn and δ-(isopropyl)D-Orn.

In another embodiment of the synthetic peptide amide of the invention:Xaa₁Xaa₂ is D-Phe-D-Phe, Xaa₃ is D-Leu or D-Nle and Xaa₄ is chosen from(B)₂D-Arg, D-Lys, (B)₂D-Nar, (B)₂D-Har, ζ-(B)D-Hlys, D-Dap,amidino-D-Dap, ε-(B)D-Lys, ε-(B)₂-D-Lys, D-Amf, amidino-D-Amf,γ-(B)₂D-Dbu and δ-(B)₂α-(B)D-Orn.

In another embodiment of the synthetic peptide amide of the invention:Xaa₄ is chosen from D-Lys, (B)₂D-Har, ε(B)-D-Lys, δ(B)₂₋α(B′)D-Orn andε(B)₂-D-Lys.

In another embodiment of the synthetic peptide amide of the invention: Gis

In another embodiment of the synthetic peptide amide of the invention:the integers p, q, r, s and t are each 1.

In another embodiment of the synthetic peptide amide of the invention:Xaa₁ is chosen from (A) D-Phe, (α-Me)D-Phe, D-Tyr, D-Tic,(tert-butyl)D-Gly, and β-(E)D-Ala, wherein A is selected from the groupconsisting of —H, —F, —Cl, —NO₂, and —CH₃, and (E) is selected from thegroup consisting of tert-butyl, cyclopentyl and thienyl; Xaa₂ isselected from the group consisting of (A)(A′)D-Phe, D-1Nal, D-2Nal,D-Tyr, and D-Trp, wherein (A′) is H or Cl; Xaa₃ is selected from thegroup consisting of D-Nle, D-Phe, (cyclopentyl)D-Ala, D-Leu,(α-Me)D-Leu, D-Hle, D-Val, and D-Met; and Xaa₄ is selected from thegroup consisting of D-Arg, (ethyl)₂D-Arg, D-Nar, D-Har, (ethyl)₂D-Har,ε-(isopropyl)D-Lys, D-Lys, D-Amf, amidino-D-Amf, β-amidino-D-Dap, D-Dbu,D-Orn, α-(methyl)D-Orn and δ-(isopropyl)D-Orn.

In another embodiment of the synthetic peptide amide of the invention:Xaa₁ is D-Phe; Xaa₂ is D-Phe; Xaa₃ is D-Leu and Xaa₄ is chosen fromD-Nar, D-Orn, and (isopropyl)D-Orn.

In another embodiment of the synthetic peptide amide of the invention: Lis a linker chosen from ε-D-Lys, ε-Lys, δ-D-Orn, δ-Orn,4-amino-4-carboxylic piperidine and bis(D-Lys-Gly)Lactam.

In another embodiment of the synthetic peptide amide, J is afive-membered heterocyclic ring moiety. In an alternative embodiment, Jis a five-membered heterocyclic ring moiety that includes twoheteroatoms, which two heteroatoms are both N.

In another embodiment of the synthetic peptide amide, G is

In a particular aspect of this embodiment, W is null, and Y is nitrogen.In another particular aspect of this embodiment, the Y and Z-containingring moiety is a five-membered saturated ring.

In another embodiment of the synthetic peptide amide, G is an optionallysubstituted proline radical.

In another embodiment of the synthetic peptide amide, the Y andZ-containing ring moiety is a six-membered saturated ring. In aparticular aspect of this embodiment, the Y and Z-containing ring moietycomprises a single heteroatom and e is zero, and R₁ and R₂ takentogether or with one or two ring atoms of the Y and Z-containing ringmoiety comprise an optionally substituted monocyclic or bicyclic 4-, 5,6-, 7, 8- or 9-membered heterocyclic ring moiety. In a particular aspectof this embodiment, R₁ and R₂ taken together with one ring atom of the Yand Z-containing ring moiety comprises a five-membered heterocyclic ringmoiety having only heteroatoms chosen from N and O, which heterocyclicring moiety with the Y and Z-containing ring moiety forms a spirostructure.

In another embodiment of the synthetic peptide amide, the Y andZ-containing ring moiety includes two heteroatoms. In a particularaspect of this embodiment, the two heteroatoms of the Y and Z-containingring moiety are both nitrogen. In another particular aspect of thisembodiment, the integer e is zero, R₂ is hydrogen and the Y- andZ-containing ring moiety is 3-substituted with R₁. In still anotherparticular aspect of this embodiment, the two heteroatoms of the Y- andZ-containing ring moiety are nitrogen and oxygen. In one particularaspect the Y- and Z-containing ring moiety is 3-substituted with R₁, theinteger e is zero and R₂ is hydrogen. In another particular aspect thetwo heteroatoms of the Y- and Z-containing ring moiety are nitrogen andsulfur. In still another particular aspect the Y- and Z-containing ringmoiety is 3-substituted with R₁, e is zero and R₂ is H.

In another embodiment of the synthetic peptide amide, G is

W is null, and Y is nitrogen and the Y and Z-containing ring moiety is aseven-membered saturated ring comprising two heteroatoms. In aparticular aspect of this embodiment, Y and Z are both nitrogen atomsand the moiety V_(e)R₁R₂ is bonded to Z. In an alternative aspect ofthis embodiment, Y is nitrogen and the second heteroatom of the Y andZ-containing ring moiety is chosen from S and O. In another alternativeaspect of this embodiment, W is —NH₂—(CH₂)_(b)— and b is zero, 1, 2, or3. In a particular aspect the Y- and Z-containing ring moiety is afive-membered saturated ring such as for instance, an optionallysubstituted proline radical. Alternatively, the Y- and Z-containing ringmoiety can be a six-membered saturated ring, wherein for example, Y canbe carbon and Z can be nitrogen; alternatively, Y and Z can both benitrogen atoms.

In another embodiment of the synthetic peptide amide, G is chosen fromsubstituted piperidinyl, piperidinyl forming a spiro structure with anoptionally substituted heterocycle, piperidinyl fused with an optionallysubstituted heterocycle, substituted piperazinyl, 4-sulfonamidylpiperazinyl, 3-substituted piperazinyl, substituted homopiperazinyl,optionally substituted homomorpholinyl, optionally substitutedhomothiomorpholinyl, 3-substituted morpholinyl, 3-substitutedthiomorpholinyl, 4-4 dioxo thiomorpholinyl, and optionally substitutedproline, and W is null; or G is

wherein the moiety

is chosen from substituted pyrazinyl, substituted pyridinyl, substitutedpiperazinyl, optionally substituted pyrimidinyl, substituted “reverse”piperidinyl (i.e. not bonded to W through the ring nitrogen), optionallysubstituted heterocyclic bicycle, optionally substituted proline,optionally substituted thiazolyl, optionally substituted dioxolanyl, andoptionally substituted tetrahydropyranyl, and W is —NH₂—(CH₂)_(b)— and bis zero, 1, 2, or 3.

In another embodiment of the synthetic peptide amide, G is chosen fromsubstituted piperidinyl, piperidinyl forming a spiro structure with anoptionally substituted heterocycle, and piperidinyl fused with anoptionally substituted heterocycle. In a particular aspect of thisembodiment, G is chosen from substituted piperazinyl, 4-sulfonamidylpiperazinyl, 3-substituted piperazinyl, and substituted homopiperazinyl.In another alternative aspect, G is chosen from optionally substitutedhomomorpholinyl, optionally substituted homothiomorpholinyl,3-substituted morpholinyl, 3-substituted thiomorpholinyl, and 4-4dioxothiomorpholinyl. In still another alternative aspect, G is anoptionally substituted proline.

In another embodiment of the synthetic peptide amide, W is—NH₂—(CH₂)_(b)—, b is zero, 1, 2, or 3 and the moiety

is chosen from optionally substituted thiazolyl, optionally substituteddioxolanyl, and optionally substituted tetrahydropyranyl. Alternatively,W is —NH₂—(CH₂)_(b)—, b is zero, 1, 2, or 3 and the moiety

is chosen from substituted pyrazinyl, substituted pyridinyl, optionallysubstituted pyrimidinyl, and optionally substituted heterocyclicbicycle. In another alternative, W is —NH₂—(CH₂)_(b)—, b is zero, 1, 2,or 3 and the moiety

is chosen from substituted piperazinyl and 4-substituted piperidinyl.

In another embodiment of the synthetic peptide amide, W is—NH₂—(CH₂)_(b)—, b is zero, 1, 2, or 3 and the moiety

is an optionally substituted proline moiety.

In another embodiment of the synthetic peptide amide, e is zero and R₁and R₂ are bonded directly to the same ring atom.

In another embodiment of the synthetic peptide amide, R₁ is chosen fromH, OH, NH₂, COOH, CH₂COOH, C₁-C₃ alkyl, amidino, C₁-C₃ alkyl-substitutedamidino, dihydroimidazole, D-Pro, D-Pro amide and CONH₂; and R₂ is H,COOH, or C₁-C₃ alkyl.

The synthetic peptide amides of the invention are useful as kappa opioidreceptor ligands and exhibit biological activity in vitro and in vivo.

A variety of assays may be employed to test whether the syntheticpeptide amides of the invention exhibit high affinity and selectivityfor the kappa opioid receptor, long duration of in vivo bioactivity, andlack of CNS side effects. Receptor assays are well known in the art andkappa opioid receptors from several species have been cloned, as have muand delta opioid receptors. Kappa opioid receptors as well as mu anddelta opioid receptors are classical, seven transmembrane-spanning, Gprotein-coupled receptors. Although these cloned receptors readily allowa particular candidate compound, e.g., a peptide or peptide derivative,to be screened, natural sources of mammalian opioid receptors are alsouseful for screening, as is well known in the art (Dooley C T et al.Selective ligands for the mu, delta, and kappa opioid receptorsidentified from a single mixture based tetrapeptide positional scanningcombinatorial library. J. Biol. Chem. 273:18848-56, 1998). Thus,screening against both kappa and mu opioid receptors, whether ofrecombinant or natural origin, may be carried out in order to determinethe selectivity of the synthetic peptide amides of the invention for thekappa over the mu opioid receptor.

In a particular embodiment, the synthetic peptide amides of theinvention are selective kappa opioid receptor agonists. The potency ofthe synthetic peptide amides of the invention as agonists for aparticular receptor can be measured as a concentration at which halfmaximal effect is achieved expressed as an EC₅₀ value. Potency of thesynthetic peptide amides of the invention as kappa opioid agonists,expressed as the percent of maximal observable effect, can be determinedby a variety of methods well known in the art. See for example, Endoh Tet al., 1999, Potent Antinociceptive Effects of TRK-820, a Novelκ-Opioid Receptor Agonist, Life Sci. 65 (16) 1685-94; and Kumar V etal., Synthesis and Evaluation of Novel Peripherally Restricted κ-OpioidReceptor Agonists, 2005 Bioorg Med Chem Letts 15: 1091-1095.

Examples of such assay techniques for determination of EC₅₀ values areprovided below. Many standard assay methods for characterization ofopioid ligands are well known to those of skill in the art. See, forexample, Waldhoer et al., (2004) Ann Rev. Biochem. 73:953-990, and Satoh& Minami (1995) Pharmac. Ther. 68(3):343-364 and references citedtherein.

In certain particular embodiments, the synthetic peptide amides of theinvention are kappa opioid receptor agonists with an EC₅₀ of less thanabout 500 nM. In other embodiments, the synthetic peptide amides have anEC₅₀ of less than about 100 nM as kappa opioid receptor agonists. Instill other embodiments, the synthetic peptide amides have an EC₅₀ ofless than about 10 nM as kappa opioid receptor agonists. In particularembodiments the synthetic peptide amides of the invention have an EC₅₀of less than about 1.0 nM, or less than about 0.1 nM, or even less thanabout 0.01 nM as kappa opioid receptor agonists. The compounds of theforegoing embodiment can have an EC₅₀ that is at least 10 times greaterfor a mu and a delta opioid receptor than for a kappa opioid receptor,preferably at least 100 times greater, and most preferably at least 1000times greater, such as for instance, an EC₅₀ of less than about 1 nM fora kappa opioid receptor, and EC₅₀ values of greater than about 1000 nMfor a mu opioid receptor and a delta opioid receptor.

In particular embodiments, the synthetic peptide amides of the inventionare highly selective for kappa over mu opioid receptors. In certainembodiments the synthetic peptide amides of the invention have EC₅₀values for the mu opioid receptor that are at least about a hundredtimes higher than the corresponding EC₅₀ values for the kappa opioidreceptor. In particular embodiments, the synthetic peptide amides of theinvention have EC₅₀ values for the mu opioid receptor that are at leastabout a thousand times higher than the corresponding EC₅₀ values for thekappa opioid receptor. Alternatively, the selectivity of the syntheticpeptide amides of the invention can be expressed as a higher EC₅₀ for amu opioid receptor than for a kappa opioid receptor. Thus, in particularembodiments, the synthetic peptide amides of the invention have EC₅₀values of greater than about 10 μM for the mu opioid receptor and EC₅₀values of less than about 10 nM, and in other embodiments less thanabout 1.0 nM, or even less than about 0.01 nM for the kappa opioidreceptor. In another embodiment, the particular synthetic peptide amidecan have an EC₅₀ of less than about 1 nM for a kappa opioid receptor andan EC₅₀ of greater than about 1000 nM for a mu opioid receptor, or for adelta opioid receptor.

Another property of the synthetic peptide amides of the invention istheir characteristic property of low inhibition of the cytochrome P₄₅₀isozymes. The cytochrome P₄₅₀ isozymes constitute a large superfamily ofhaem-thiolate proteins responsible for metabolic oxidative inactivationof many therapeutics and other bioactive compounds. Usually, they act asterminal oxidases in multicomponent electron transfer chains, alsoreferred to as cytochrome P₄₅₀-containing monooxygenase systems.

Over fifty different cytochrome P₄₅₀ isozymes have been identified andhave been classified into families grouped by genetic relatedness asassessed by nucleic acid sequence homology. Most abundant among thecytochrome P₄₅₀ isozymes in human cells are the 1A2 and 3A4 isozymes,although isozymes 2B6, 2C9, 2C19, 2D6, and 2E1 also contributesignificantly to oxidative inactivation of administered therapeutics.While inhibition of the cytochrome P₄₅₀ isozymes may be useful inprolonging the time after in vivo administration during which aneffective concentration of the synthetic peptide amides of the inventionis maintained, it also prolongs the persistence of any co-administeredtherapeutic compound that is subject to oxidation by cytochrome P₄₅₀.This increase in persistence may cause the co-administered therapeuticto persist beyond the period that is optimal for therapy, or may causethe in vivo concentration to exceed the desired levels or safelytolerated levels. Such increases in persistence and/or increases inconcentration are difficult to accurately quantify and are preferablyavoided. Therapeutics that show little or no inhibition of the activityof the cytochrome P₄₅₀ isozymes do not have this potential problem andcan be more safely co-administered with other therapeutics without riskof affecting the rate of inactivation of the co-administered therapeuticcompound by the cytochrome P₄₅₀ isozymes.

Particular embodiments of the synthetic peptide amides of the inventionshow low inhibition of the cytochrome P₄₅₀ isozymes at therapeuticconcentrations of the synthetic peptide amides, while others showessentially no inhibition of the cytochrome P₄₅₀ isozymes at therapeuticconcentrations. In some embodiments, the synthetic peptide amides at aconcentration of 10 μM show less than about 50% inhibition of cytochromeP₄₅₀ isozymes CYP1A2, CYP2C9, CYP2C19 or CYP2D6. In particularembodiments, the synthetic peptide amides at a concentration of 10 μMshow less than about 20% inhibition of any of these cytochrome P₄₅₀isozymes. In very particular embodiments, the synthetic peptide amidesat a concentration of 10 μM show less than about 10% inhibition of anyof these cytochrome P₄₅₀ isozymes.

In another embodiment, the synthetic peptide amides of the invention atan effective concentration exhibit no more than about 50% inhibition ofany of P₄₅₀ CYP1A2, CYP2C9, CYP2C19 or CYP 2D6 by the synthetic peptideamide at a concentration of 10 μM after 60 minutes incubation with humanliver microsomes.

The synthetic peptide amides of the invention when administered to amammal or a human patient at a therapeutically effective concentrationexhibit low or essentially no penetration across the blood-brainbarrier. Kappa opioid receptors (hereinafter interchangeably referred toas kappa receptors) are distributed in peripheral tissues, including theskin and somatic tissues, as well as the viscera in humans and othermammals. Kappa receptors are also found in the brain. Activation of thekappa receptors in peripheral tissues causes suppression of pain andinflammatory responses, while activation of the kappa receptors in thebrain causes sedative effects and may also lead to severe dysphoria andhallucinations. In certain embodiments, the synthetic peptide amides ofthe invention when administered at therapeutically effectiveconcentrations exhibit little or essentially no penetration across theblood-brain barrier and therefore minimize or even completely obviatethe sedative, hallucinogenic effects of many other kappa agonists thatshow some penetration across the blood-brain bather.

One useful measure of the extent to which the synthetic peptide amidesof the invention cross the blood-brain barrier is the ratio of the peakplasma concentration to the concentration in brain tissue. In particularembodiments, the synthetic peptide amides of the invention whenadministered at a dose of about 3 mg/kg, exhibit at least about a fivefold lower peak concentration of the synthetic peptide amide in brainthan the peak concentration in plasma.

Another useful measure of the extent to which the synthetic peptideamides of the invention cross the blood-brain barrier is the ratio ofthe dose required to achieve a sedative effect and the dose required toachieve an analgesic effect. The analgesic and sedative effects of kappareceptor stimulation by kappa receptor agonists can be measured bystandard assays well known to those of skill in the art.

In particular embodiments, the synthetic peptide amides of the inventionhave an ED₅₀ for a sedative effect that is at least about ten times theED₅₀ for an analgesic effect. In particular embodiments, the syntheticpeptide amides of the invention have an ED₅₀ for a sedative effect thatis at least about thirty times the ED₅₀ for an analgesic effect. Instill other embodiments, the synthetic peptide amides of the inventionhave an ED₅₀ for a sedative effect that is at least about fifty timesthe ED₅₀ for an analgesic effect.

In one aspect, the synthetic peptide amide of the invention has an ED₅₀for a sedative effect in a locomotion-reduction assay in a mouse atleast about ten times the ED₅₀ of the synthetic peptide amide for ananalgesic effect in a writhing assay in a mouse.

Another useful predictor of the extent to which the synthetic peptideamides of the invention would be expected to cross the blood-brainbarrier is provided by the membrane permeability values of the syntheticpeptide amides into a human cell or other mammalian cell when deliveredat a therapeutically relevant concentration. In certain embodiments, thesynthetic peptide amides of the invention at therapeutically relevantconcentrations exhibit low or essentially no ability to penetrate amonolayer of suitably cultured human or other mammalian cells. Thispermeability parameter can be expressed as an apparent permeability,P_(app), representing the permeability of the particular cell monolayerto a compound of interest. Any suitably culturable mammalian cellmonolayer can be used to determine its permeability for a particularcompound of interest, although certain cell lines are frequently usedfor this purpose. For instance, the Caco-2 cell line is a human colonadenocarcinoma that can be used as a monolayer culture test system fordetermination of membrane permeability towards compounds of theinvention. In certain embodiments, the synthetic peptide amides of theinvention have a P_(app) of less than about 10⁻⁶ cm/sec. In certainother embodiments, the synthetic peptide amides of the invention have aP_(app) of less than about 10⁻⁷ cm/sec.

In one embodiment, the synthetic peptide amide of the invention at adose of about 3 mg/kg in rat reaches a peak plasma concentration andexhibits at least about a five fold lower peak concentration in brainthan such peak plasma concentration.

In another embodiment, the synthetic peptide amides of the inventionhave at least about 50% of maximum efficacy at about 3 hours postadministration of a dose of about 3 mg/kg of the synthetic peptide amidein a rat.

In one embodiment the synthetic peptide amide of the invention exhibitsa long lasting duration of action in a mammal, such as a human. In oneaspect, the synthetic peptide amide has a duration of action that is atleast about 50% of maximum efficacy at three hours post administrationof 0.1 mg/kg of the synthetic peptide amide. In another aspect thesynthetic peptide amide has a duration of action that is at least about75% of maximum efficacy at three hours post administration of 0.1 mg/kgof the synthetic peptide amide. In a particular aspect the syntheticpeptide amide has a duration of action that is at least about 90% ofmaximum efficacy at 3hrs post administration of 0.1 mg/kg of thesynthetic peptide amide. In a specific aspect, the synthetic peptideamide has a duration of action that is at least about 95% of maximumefficacy at three hours post administration of 0.1 mg/kg of thesynthetic peptide amide.

In another embodiment, the invention provides a pharmaceuticalcomposition that includes a synthetic peptide amide according to any ofthe above embodiments and a pharmaceutically acceptable excipient orcarrier. The invention provides methods, compositions, or dosage formsthat include synthetic peptide amides of the invention that areselective for the kappa opioid receptor. In particular embodiments, thesynthetic peptide amides of the invention exhibit a strong affinity forthe kappa opioid receptor and have a high potency as kappa opioidreceptor agonists.

A pro-drug of a compound such as the synthetic peptide amides of theinvention include pharmaceutically acceptable derivatives which uponadministration can convert through metabolism or other process to abiologically active form of the compound. Pro-drugs are particularlydesirable where the pro-drug has more favorable properties than does theactive compound with respect to bioavailability, stability orsuitability for a particular formulation.

As used herein, a kappa opioid receptor-associated disease or conditionis any disease, condition or disorder that is preventable or treatableby activation of a kappa opioid receptor. In one aspect, the syntheticpeptide amides of the invention are kappa opioid receptor agonists thatactivate the kappa opioid receptor. In some embodiments, a particulardose and route of administration of the synthetic peptide amide of theinvention can be chosen by a clinician to completely prevent or cure thedisease, condition or disorder. In other embodiments a particular doseand route of administration of the synthetic peptide amide of theinvention chosen by the clinician ameliorates or reduces one or moresymptoms of the disease, condition or disorder.

As used herein, “effective amount” or “sufficient amount” of thesynthetic peptide amide of the invention refers to an amount of thecompound as described herein that may be therapeutically effective toinhibit, prevent, or treat a symptom of a particular disease, disorder,condition, or side effect. As used herein, a “reduced dose” of a muopioid agonist analgesic compound refers to a dose which when used incombination with a kappa opioid agonist, such as a synthetic peptideamide of the invention, is lower than would be ordinarily provided to aparticular patient, for the purpose of reducing one or more side effectsof the compound. The dose reduction can be chosen such that the decreasein the analgesic or other therapeutic effect of the compound is anacceptable compromise in view of the reduced side effect(s), where thedecrease in analgesic or other therapeutic effects of the mu opioidagonist analgesic are wholly or at least partially offset by theanalgesic or other therapeutic effect of the synthetic peptide amide ofthe invention. Co-administration of a mu opioid agonist analgesiccompound with a synthetic peptide amide of the invention which acts as akappa opioid agonist also permits incorporation of a reduced dose of thesynthetic peptide amide and/or the mu opioid agonist analgesic compoundto achieve the same therapeutic effect as a higher dose of the syntheticpeptide amide or the mu opioid agonist analgesic compound ifadministered alone.

As used herein, “pharmaceutically acceptable” refers to compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for contact with the tissues ofhuman beings and animals without severe toxicity, irritation, allergicresponse, or other complications, commensurate with a benefit-to-riskratio that is reasonable for the medical condition being treated.

As used herein, “dosage unit” refers to a physically discrete unitsuited as unitary dosages for a particular individual or condition to betreated. Each unit may contain a predetermined quantity of activesynthetic peptide amide compound(s) calculated to produce the desiredtherapeutic effect(s), optionally in association with a pharmaceuticalcarrier. The specification for the dosage unit forms may be dictated by(a) the unique characteristics of the active compound or compounds, andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such active compound orcompounds. The dosage unit is often expressed as weight of compound perunit body weight, for instance, in milligrams of compound per kilogramof body weight of the subject or patient (mg/kg). Alternatively, thedosage can be expressed as the amount of the compound per unit bodyweight per unit time, (mg/kg/day) in a particular dosage regimen. In afurther alternative, the dosage can be expressed as the amount ofcompound per unit body surface area (mg/m²) or per unit body surfacearea per unit time (mg/m²/day). For topical formulations, the dosage canbe expressed in a manner that is conventional for that formulation,e.g., a one-half inch ribbon of ointment applied to the eye, where theconcentration of compound in the formulation is expressed as apercentage of the formulation.

As used herein, a “pharmaceutically acceptable salt” refers to aderivative of a compound wherein the parent compound is modified bymaking an acid or a base salt thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For instance,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric acids and the like; and the salts prepared from organic acidssuch as acetic, propionic, succinic, glycolic, stearic, lactic, malic,tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionicacids, and the like. These physiologically acceptable salts are preparedby methods known in the art, e.g., by dissolving the free amine baseswith an excess of the acid in aqueous alcohol, or neutralizing a freecarboxylic acid with an alkali metal base such as a hydroxide, or withan amine. Thus, a pharmaceutically acceptable salt of a syntheticpeptide amide can be formed from any such peptide amide having eitheracidic, basic or both functional groups. For example, a peptide amidehaving a carboxylic acid group, may in the presence of apharmaceutically suitable base, form a carboxylate anion paired with acation such as a sodium or potassium cation. Similarly, a peptide amidehaving an amine functional group may, in the presence of apharmaceutically suitable acid such as HCl, form a salt.

An example of a pharmaceutically acceptable solvate of a syntheticpeptide amide is a combination of a peptide amide with solvent moleculeswhich yields a complex of such solvent molecules in association with thepeptide amide. Combinations of a drug and propyleneglycol(1,2-propanediol) have been used to form pharmaceutical drugsolvates. See for example U.S. Pat. No. 3,970,651. Other suitablesolvates are hydrates of drug compounds. Such hydrates include hydrateswhich either have comparable activity or hydrates which are convertedback to the active compound following administration. A pharmaceuticallyacceptable N-oxide of a synthetic peptide amide is such a compound thatcontains an amine group wherein the nitrogen of the amine is bonded toan oxygen atom.

A pharmaceutically acceptable crystalline, isomorphic crystalline oramorphous form of a synthetic peptide amide of the invention can be anycrystalline or non-crystalline form of a pharmaceutically acceptableacidic, basic, zwitterionic, salt, hydrate or any other suitably stable,physiologically compatible form of the synthetic peptide amide accordingto the invention.

The synthetic peptide amides of the invention can be incorporated intopharmaceutical compositions. The compositions can include an effectiveamount of the synthetic peptide amide in a pharmaceutically acceptablediluent, excipient or carrier. Conventional excipients, carriers and/ordiluents for use in pharmaceutical compositions are generally inert andmake up the bulk of the preparation.

In a particular embodiment, the synthetic peptide amide is a kappaopioid receptor agonist. In another embodiment, the synthetic peptideamide is a selective kappa opioid receptor agonist. The target site canbe a kappa receptor in the patient or subject in need of such treatmentor prophylaxis. Certain synthetic peptide amide kappa opioid receptoragonists of the invention are peripherally acting and show little or noCNS effects at therapeutically effective doses.

The pharmaceutical excipient or carrier can be any compatible, non-toxicsubstance suitable as a vehicle for delivery the synthetic peptide amideof the invention. Suitable excipients or carriers include, but are notlimited to, sterile water (preferably pyrogen-free), saline,phosphate-buffered saline (PBS), water/ethanol, water/glycerol,water/sorbitol, water/polyethylene glycol, propylene glycol,cetylstearyl alcohol, carboxymethylcellulose, corn starch, lactose,glucose, microcrystalline cellulose, magnesium stearate,polyvinylpyrrolidone (PVP), citric acid, tartaric acid, oils, fattysubstances, waxes or suitable mixtures of any of the foregoing.

The pharmaceutical composition according to the invention can beformulated as a liquid, semisolid or solid dosage form. For example thepharmaceutical preparation can be in the form of a solution forinjection, drops, syrup, spray, suspension, tablet, patch, capsule,dressing, suppository, ointment, cream, lotion, gel, emulsion, aerosolor in a particulate form, such as pellets or granules, optionallypressed into tablets or lozenges, packaged in capsules or suspended in aliquid. The tablets can contain binders, lubricants, diluents, coloringagents, flavoring agents, wetting agents and may be enteric-coated tosurvive the acid environment of the stomach and dissolve in the morealkaline conditions of the intestinal lumen. Alternatively, the tabletscan be sugar-coated or film coated with a water-soluble film.Pharmaceutically acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions.

Binders include for instance, starch, mucilage, gelatin and sucrose.Lubricants include talc, lycopodium, magnesium and calciumstearate/stearic acid. Diluents include lactose, sucrose, mannitol,salt, starch and kaolin. Wetting agents include propylene glycol andsorbitan monostearate.

As used herein, local application or administration refers toadministration of a pharmaceutical preparation according to theinvention to the site, such as an inflamed joint, that exhibits thepainful and/or inflamed condition. Such local application includesintrajoint, such as intra-articular application, via injection,application via catheter or delivery as part of a biocompatible device.Thus, local application refers to application to a discrete internalarea of the body, such as, for example, a joint, soft tissue area (suchas muscle, tendon, ligaments, intraocular or other fleshy internalareas), or other internal area of the body. In particular, as usedherein, local application refers to applications that providesubstantially no systemic delivery and/or systemic administration of theactive agents in the present compositions. Also, as used herein, localapplication is intended to refer to applications to discrete areas ofthe body, that is, other than the various large body cavities (such as,for example, the peritoneal and/or pleural cavities).

As used herein, topical application refers to application to the surfaceof the body, such as to the skin, eyes, mucosa and lips, which can be inor on any part of the body, including but not limited to the epidermis,any other dermis, or any other body tissue. Topical administration orapplication means the direct contact of the pharmaceutical preparationaccording to the invention with tissue, such as skin or membrane,particularly the cornea, or oral, vaginal or anorectal mucosa. Thus, forpurposes herein topical application refers to application to the tissueof an accessible body surface, such as, for example, the skin (the outerintegument or covering) and the mucosa (the mucus-producing, secretingand/or containing surfaces). In particular, topical application refersto applications that provide little or substantially no systemicdelivery of the active compounds in the present compositions. Exemplarymucosal surfaces include the mucosal surfaces of the eyes, mouth (suchas the lips, tongue, gums, cheeks, sublingual and roof of the mouth),larynx, esophagus, bronchus, trachea, nasal passages, vagina andrectum/anus.

For oral administration, an active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, or entericcoated for selective disintegration in the gastrointestinal tract.Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. To facilitate drug stabilityand absorption, peptides of the invention can be released from a capsuleafter passing through the harsh proteolytic environment of the stomach.Methods for enhancing peptide stability and absorption after oraladministration are well known in the art (e.g., Mahato R I. Emergingtrends in oral delivery of peptide and protein drugs. Critical Reviewsin Therapeutic Drug Carrier Systems. 20:153-214, 2003).

Dosage forms such as lozenges, chewable tablets and chewing gum permitmore rapid therapeutic action compared to per-oral dosage forms of thesynthetic peptide amide compounds of the invention having significantbuccal absorption. Chewing gum formulations are solid, single dosepreparations with a base consisting mainly of gum, that are intended tobe chewed but not swallowed, and contain one or more compounds of theinvention which are released by chewing and are intended to be used forlocal treatment of pain and inflammation of the mouth or systemicdelivery after absorption through the buccal mucosa. See for example,U.S. Pat. No. 6,322,828 to Athanikar and Gubler entitled: Process formanufacturing a pharmaceutical chewing gum.

For nasal administration, the peripherally selective kappa opioidreceptor agonists can be formulated as aerosols. The term “aerosol”includes any gas-borne suspended phase of the compounds of the instantinvention which is capable of being inhaled into the bronchioles ornasal passages. Specifically, aerosol includes a gas-borne suspension ofdroplets of the compounds of the instant invention, as may be producedin a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosolalso includes a dry powder composition of a compound of the instantinvention suspended in air or other carrier gas, which may be deliveredby insufflation from an inhaler device, for example. See Ganderton &Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987);Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems6:273-313; and Raeburn et al. (1992) J. Pharmacol. Toxicol. Methods27:143-159.

The pharmaceutical compositions of the invention can be prepared in aformulation suitable for systemic delivery, such as for instance byintravenous, subcutaneous, intramuscular, intraperitoneal, intranasal,transdermal, intravaginal, intrarectal, intrapulmonary or oral delivery.Alternatively, the pharmaceutical compositions of the invention can besuitably formulated for local delivery, such as, for instance, fortopical, or iontophoretic delivery, or for transdermal delivery by apatch coated, diffused or impregnated with the formulation, and localapplication to the joints, such as by intra-articular injection.

Preparations for parenteral administration include sterile solutionsready for injection, sterile dry soluble products ready to be combinedwith a solvent just prior to use, including hypodermic tablets, sterilesuspensions ready for injection, sterile dry insoluble products ready tobe combined with a vehicle just prior to use and sterile emulsions. Thesolutions may be either aqueous or nonaqueous, and thereby formulatedfor delivery by injection, infusion, or using implantable pumps. Forintravenous, subcutaneous, and intramuscular administration, usefulformulations of the invention include microcapsule preparations withcontrolled release properties (R. Pwar et al. Protein and peptideparenteral controlled delivery. Expert Opin Biol Ther. 4(8):1203-12,2004) or encapsulation in liposomes, with an exemplary form beingpolyethylene coated liposomes, which are known in the art to have anextended circulation time in the vasculature (e.g. Koppal, T. “Drugdelivery technologies are right on target”, Drug Discov. Dev. 6, 49-50,2003).

For ophthalmic administration, the present invention provides a methodof treating glaucoma or ophthalmic pain and inflammation, comprisingadministering to an eye of a patient in need thereof a therapeuticallyeffective amount of a synthetic peptide amide of the invention. Thesynthetic peptide amide can be administered topically with aneye-compatible pharmaceutical carrier or non-systemically using acontact lens or intraocular implant that can optionally contain polymersthat provide sustained release of the synthetic peptide amide. Sucheye-compatible pharmaceutical carriers can include adjuvants,antimicrobial preservatives, surfactants, and viscolyzers etc. It isknown in the art that high concentrations of many compounds are irritantto the eye and low concentrations are less irritant; thus theformulation is often designed to include the lowest effectiveconcentrations of active compound, preservative, surfactant, and/orviscolyzer, said viscolyzer preferably having a high surface tension toreduce irritation of the eye while increasing the retention ofophthalmic solutions at the eye surface. Such controlled release of thesynthetic peptide amides of the invention can last 6 months to a yearfor implants, or for shorter periods (3-14 days) for contact lenses.Such implants can be osmotic pumps, biodegradable matrices, orintraocular sustained release devices. Such topical compositions caninclude a buffered saline solution with or without liposomes.

Aqueous polymeric solutions, aqueous suspensions, ointments, and gelscan be used for topical formulations of the synthetic peptide amides ofthe invention for ocular applications. The aqueous formulations may alsocontain liposomes for creating a reservoir of the synthetic peptideamide. Certain of these topical formulations are gels which enhancepre-corneal retention without the inconvenience and impairment of visionassociated with ointments. The eye-compatible pharmaceutical carrier canalso include a biodegradable synthetic polymer. Biodegradablemicrosphere compositions approved for human use include thepolylactides: poly(lactic acid), poly(glycolic acid), andpoly(lactic-coglycolic) acid. Additional biodegradable formulationsinclude, but are not limited to: poly(anhydride-co-imide),poly(lactic-glycolic acid), polyethyl-2-cyanoacrylate, polycaprolactone,polyhydroxybutyrate valerate, polyorthoester, andpolyethylene-oxide/polybutylene teraphthalate. Intraocular implantationor injection of sustained release compositions that include a syntheticpeptide amide of the invention can provide long-term control (rangingfrom months to years) of intraocular pressure, and thereby avoiding orreducing the need for topical preparations. Useful methods forformulating and dispensing ophthalmic medications are disclosed in U.S.Pat. No. 7,122,579 to Schwartz et al, and in U.S. Pat. No. 7,105,512 toMorizono et al. Methods for formulating ophthalmic medications incontact lenses are disclosed by Gulsen and Chauhan, Ophthalmic drugdelivery through contact lenses. Investigative Ophthalmology and VisualScience, (2004) 45:2342-2347.

Preparations for transdermal delivery are incorporated into a devicesuitable for said delivery, said device utilizing, e.g., iontophoresis(Kalia Y N et al. Iontophoretic Drug Delivery. Adv Drug Deliv Rev.56:619-58, 2004) or a dermis penetrating surface (Prausnitz M R.Microneedles for Transdermal Drug Delivery. Adv Drug Deliv Rev.56:581-7, 2004), such as are known in the art to be useful for improvingthe transdermal delivery of drugs. An electrotransport device andmethods of operation thereof are disclosed in U.S. Pat. No. 6,718,201.Methods for the use of iontophoresis to promote transdermal delivery ofpeptides are disclosed in U.S. Pat. Nos. 6,313,092 and 6,743,432.

As used herein the terms “electrotransport”, “iontophoresis”, and“iontophoretic” refer to the delivery through a body surface (e.g., skinor mucosa) of one or more pharmaceutically active compounds by means ofan applied electromotive force to an agent containing reservoir. Thecompound may be delivered by electromigration, electroporation,electroosmosis or any combination thereof. Electroosmosis has also beenreferred to as electrohydrokinesis, electro convection, and electricallyinduced osmosis. In general, electroosmosis of a compound into a tissueresults from the migration of solvent in which the compound iscontained, as a result of the application of electromotive force to thetherapeutic species reservoir, such as for instance, solvent flowinduced by electromigration of other ionic species. During theelectrotransport process, certain modifications or alterations of theskin may occur such as the formation of transiently existing pores inthe skin, also referred to as “electroporation.” Any electricallyassisted transport of species enhanced by modifications or alterationsto the body surface (e.g., formation of pores in the skin) are alsoincluded in the term “electrotransport” as used herein. Thus, as usedherein, applied to the compounds of the instant invention, the terms“electrotransport”, “iontophoresis” and “iontophoretic” refer to (1) thedelivery of charged agents by electromigration, (2) the delivery ofuncharged agents by the process of electroosmosis, (3) the delivery ofcharged or uncharged agents by electroporation, (4) the delivery ofcharged agents by the combined processes of electromigration andelectroosmosis, and/or (5) the delivery of a mixture of charged anduncharged agents by the combined processes of electromigration andelectroosmosis. Electrotransport devices generally employ twoelectrodes, both of which are positioned in close electrical contactwith some portion of the skin of the body. One electrode, called theactive or donor electrode, is the electrode from which the therapeuticagent is delivered into the body. The other electrode, called thecounter or return electrode, serves to close the electrical circuitthrough the body. In conjunction with the patient's skin, the circuit iscompleted by connection of the electrodes to a source of electricalenergy, e.g., a battery, and usually to circuitry capable of controllingcurrent passing through the device.

Depending upon the electrical charge of the compound to be deliveredtransdermally, either the anode or cathode may be the active or donorelectrode. Thus, if the compound to be transported is positivelycharged, e.g., the compound exemplified in Example 1 herein, then thepositive electrode (the anode) will be the active electrode and thenegative electrode (the cathode) will serve as the counter electrode,completing the circuit. However, if the compound to be delivered isnegatively charged, then the cathodic electrode will be the activeelectrode and the anodic electrode will be the counter electrode.Electrotransport devices additionally require a reservoir or source ofthe therapeutic agent that is to be delivered into the body. Such drugreservoirs are connected to the anode or the cathode of theelectrotransport device to provide a fixed or renewable source of one ormore desired species or agents. Each electrode assembly is comprised ofan electrically conductive electrode in ion-transmitting relation withan ionically conductive liquid reservoir which in use is placed incontact with the patient's skin. Gel reservoirs such as those describedin Webster (U.S. Pat. No. 4,383,529) are one form of reservoir sincehydrated gels are easier to handle and manufacture than liquid-filledcontainers. Water is one liquid solvent that can be used in suchreservoirs, in part because the salts of the peptide compounds of theinvention are water soluble and in part because water is non-irritatingto the skin, thereby enabling prolonged contact between the hydrogelreservoir and the skin. For electrotransport, the synthetic peptides ofthe invention can be formulated with flux enhancers such as ionicsurfactants or cosolvents other than water (See for example, U.S. Pat.No. 4,722,726 and European Patent Application 278,473, respectively).Alternatively the outer layer (i.e., the stratum corneum) of the skincan be mechanically disrupted prior to electrotransport deliverytherethrough, for example as described in U.S. Pat. No. 5,250,023.

Peripherally synthetic peptide amides that are well suited forelectrotransport can be selected by measuring their electrotransportflux through the body surface (e.g., the skin or mucosa), e.g., ascompared to a standardized test peptide with known electrotransport fluxcharacteristics, e.g. thyrotropin releasing hormone (R. Burnette et al.J. Pharm. Sci. (1986) 75:738) or vasopressin (Nair et al. Pharmacol Res.48:175-82, 2003). Transdermal electrotransport flux can be determinedusing a number of in vivo or in vitro methods well known in the art. Invitro methods include clamping a piece of skin of an appropriate mammal(e.g., human cadaver skin) between the donor and receptor compartmentsof an electrotransport flux cell, with the stratum corneum side of theskin piece facing the donor compartment. A liquid solution or gelcontaining the drug to be delivered is placed in contact with thestratum corneum, and electric current is applied to electrodes, oneelectrode in each compartment. The transdermal flux is calculated bysampling the amount of drug in the receptor compartment. Two successfulmodels used to optimize transdermal electrotransport drug delivery arethe isolated pig skin flap model (Heit M C et al. Transdermaliontophoretic peptide delivery: in vitro and in vivo studies withluteinizing hormone releasing hormone. J. Pharm. Sci. 82:240-243, 1993),and the use of isolated hairless skin from hairless rodents or guineapigs, for example. See Hadzija B W et al. Effect of freezing oniontophoretic transport through hairless rat skin. J. Pharm. Pharmacol.44, 387-390, 1992. Compounds of the invention for transdermaliontophoretic delivery can have one, or typically, two chargednitrogens, to facilitate their delivery.

Other useful transdermal delivery devices employ high velocity deliveryunder pressure to achieve skin penetration without the use of a needle.Transdermal delivery can be improved, as is known in the art, by the useof chemical enhancers, sometimes referred to in the art as “permeationenhancers”, i.e., compounds that are administered along with the drug(or in some cases used to pretreat the skin, prior to drugadministration) in order to increase the permeability of the stratumcorneum, and thereby provide for enhanced penetration of the drugthrough the skin. Chemical penetration enhancers are compounds that areinnocuous and serve merely to facilitate diffusion of the drug throughthe stratum corneum, whether by passive diffusion or an energy drivenprocess such as electrotransport. See, for example, Meidan V M et al.Enhanced iontophoretic delivery of buspirone hydrochloride across humanskin using chemical enhancers. Int. J. Pharm. 264:73-83, 2003.

Pharmaceutical dosage forms for rectal administration include rectalsuppositories, capsules and tablets for systemic effect. Rectalsuppositories as used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesinclude bases or vehicles and agents that raise the melting point of thesuppositories. Examples of bases include cocoa butter (theobroma oil),glycerin-gelatin, carbowax, (polyoxyethylene glycol) and appropriatemixtures of mono-, di- and triglycerides of fatty acids. Combinations ofthe various bases can also be used. Agents that raise the melting pointof suppositories include spermaceti and wax. Rectal suppositories may beprepared either by the compression method or by molding. Rectalsuppositories typically weigh about 2 gm to about 3 gm. Tablets andcapsules for rectal administration are manufactured using the samepharmaceutically acceptable substance(s) and by the same methods as forformulations for oral administration.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include sodium chloride for injection,Ringers solution for injection, isotonic dextrose for injection, sterilewater for injection, dextrose and lactated Ringers solution forinjection. Nonaqueous parenteral vehicles include fixed oils ofvegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.Antimicrobial agents in bacteriostatic or fungistatic concentrationsmust be added to parenteral preparations packaged in multiple dosecontainers which include phenols or cresols, mercurials, benzyl alcohol,chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters,thimerosal, benzalkonium chloride and benzethonium chloride. Isotonicagents include sodium chloride and dextrose. Buffers include phosphateand citrate. Antioxidants include sodium bisulfite. Local anestheticsinclude procaine hydrochloride. Suspending and dispersing agents includesodium carboxymethylcelluose, hydroxypropyl methylcellulose andpolyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (Tween80). A sequestering or chelating agent of metal ions such as EDTA canalso be incorporated. Pharmaceutical carriers also include ethylalcohol, polyethylene glycol and propylene glycol for water misciblevehicles and the pH can be adjusted to a physiologically compatible pHby addition of sodium hydroxide, hydrochloric acid, citric acid orlactic acid.

The active ingredient may be administered all at once, or may be dividedinto a number of smaller doses to be administered at intervals of time,or as a controlled release formulation. The term “controlled releaseformulation” encompasses formulations that allow the continuous deliveryof a synthetic peptide amide of the invention to a subject over a periodof time, for example, several days to weeks. Such formulations may beadministered subcutaneously or intramuscularly and allow for thecontinual steady state release of a predetermined amount of compound inthe subject over time. The controlled release formulation of syntheticpeptide amide may be, for example, a formulation of drug containingpolymeric microcapsules, such as those described in U.S. Pat. Nos.4,677,191 and 4,728,721, incorporated herein by reference. Theconcentration of the pharmaceutically active compound is adjusted sothat administration provides an effective amount to produce a desiredeffect. The exact dose depends on the age, weight and condition of thepatient or animal, as is known in the art. For any particular subject,specific dosage regimens can be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the formulations.Thus, the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedinvention.

The unit dose parenteral preparations include packaging in an ampoule orprepackaged in a syringe with, or without a needle for delivery. Allpreparations for parenteral administration are typically sterile, as ispracticed in the art. Illustratively, intravenous infusion of a sterileaqueous buffered solution containing an active compound is an effectivemode of administration. In another embodiment a sterile aqueous or oilysolution or suspension containing the active material can be injected asnecessary to produce the desired pharmacological effect.

The pharmaceutical compositions of the invention can be delivered oradministered intravenously, transdermally, transmucosally, intranasally,subcutaneously, intramuscularly, orally or topically (such as forexample to the eye). The compositions can be administered forprophylaxis or treatment of individuals suffering from, or at risk of adisease or a condition. Prophylaxis is defined as a measure designed topreserve the health of an individual. For therapeutic applications, apharmaceutical composition is typically administered to a subjectsuffering from a disease or condition, in an amount sufficient toinhibit, prevent, or ameliorate the disease or condition. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.”

The pharmaceutical compositions of the invention can be administered toa mammal for prophylactic or therapeutic purposes in any of theabove-described formulations and delivery modes. The mammal can be anymammal, such as a domesticated or feral mammal, or even a wild mammalThe mammal can be any primate, ungulate, canine or feline. For instance,and without limitation, the mammal may be a pet or companion animal,such as a dog or a cat; a high-value mammal such as a thoroughbred horseor a show animal; a farm animal, such as a cow, a goat, a sheep or pig;or a primate such as an ape, gorilla, orangutan, lemur, monkey orchimpanzee. Humans are suitable mammals for prophylaxis or treatmentusing pharmaceutical compositions of the invention.

The pharmaceutical compositions of the invention can be administered toa mammal having a disease or condition treatable by activation of thekappa opioid receptor. Alternatively, the pharmaceutical compositionscan be administered as prophylactics to a mammal having a risk ofcontracting or developing a disease or condition preventable byactivation of the kappa opioid receptor. Diseases or conditions that canbe treated or prevented by administration of the pharmaceuticalcompositions of the invention include, without limitation, any conditionthat can be ameliorated by activation of the kappa opioid receptor,including such conditions as pain, inflammation, pruritis, hyponatremia,hypokalemia, congestive heart failure, liver cirrhosis, nephroticsyndrome, hypertension, edema, ileus, tussis and glaucoma.

In a particular embodiment, the pharmaceutical compositions of theinvention can be co-administered with or can include one or more othertherapeutic compounds or adjuvants, such as but not limited to otheropioids, cannabinoids, antidepressants, anticonvulsants, neuroleptics,antihistamines, acetaminophen, corticosteroids, ion channel blockingagents, non-steroidal anti-inflammatory drugs (NSAIDs), and diuretics,many of which are synergistic in effect with the synthetic peptideamides of the invention.

Suitable opioids, include, without limitation, alfentanil, alphaprodine,anileridine, bremazocine, buprenorphine, butorphanol, codeine,conorphone, dextromoramide, dextropropoxyphene, dezocine, diamorphine,dihydrocodeine, dihydromorphine, diphenoxylate, dipipanone, doxpicomine,ethoheptazine, ethylketazocine, ethylmorphine, etorphine, fentanyl,hydrocodone, hydromorphone, ketobemidone, levomethadyl, levorphanol,lofentanil, loperamide, meperidine(pethidine), meptazinol, methadone,morphine, morphine-6-glucuronide, nalbuphine, nalorphine, nicomorphine,oxycodone, oxymorphone, pentazocine, phenazocine, phenoperidine,piritramide, propiram, propoxyphene, remifentanil, sufentanil, tilidate,tonazocine, and tramadol.

One embodiment of the invention is co-formulation and/orco-administration of an opioid with substantial agonist activity at themu opioid receptor, such as morphine, fentanyl, hydromorphone, oroxycodone, together with a synthetic peptide amide of the invention, forthe purpose of a mu opioid dose-sparing effect, where the dose of the muopioid is reduced to minimize common mu opioid side effects,particularly in opioid-naive patients. Such side effects includeconstipation, nausea, vomiting, sedation, respiratory depression,pruritis (itching), mental confusion, disorientation and cognitiveimpairment, urinary retention, biliary spasm, delirium, myoclonic jerks,and seizures. The selection of the reduced mu opioid dose requiresexpert clinical judgment, and depends on the unique characteristics ofthe various mu opioids, as well as patient characteristics such as painintensity, patient age, coexisting disease, current drug regimen andpotential drug interactions, prior treatment outcomes, and patientpreference (McCaffery, M. and Pasero, C., Pain Clinical Manual, SecondEdition, Mosby, 1999).

Cannabinoids suitable for administration with or incorporation into thepharmaceutical compositions of the invention, include any naturalcannabinoid, such as for instance, tetrahydrocannabinol (THC), or a THCderivative, or a synthetic cannabinoid, such as, for instance,levonantradol, marinol, nabilone, rimonabant or savitex.

Suitable antidepressants that can be co-administered with orincorporated into the pharmaceutical compositions of the invention,include for example, tricyclic antidepressants such as imipramine,desipramine, trimipramine, protriptyline, norttriptyline, amitriptyline,doxepin, and clomipramine; atypical antidepressants such as amoxapine,maprotiline, trazodone, bupropion, and venlafaxine; serotonin-specificreuptake inhibitors such as fluoxetine, sertraline, paroxetine,citalopram and fluvoxamine; norepinephrine-specific reuptake inhibitorssuch as reboxetine; or dual-action antidepressants such as nefazodoneand mirtazapine.

Suitable neuroleptics that can be co-administered with or incorporatedinto the pharmaceutical compositions of the invention, include anyneuroleptic, for example, a compound with D2 dopamine receptorantagonist activity such as domperidone, metaclopramide, levosulpiride,sulpiride, thiethylperazine, ziprasidone, zotepine, clozapine,chlorpromazine, acetophenazine, carphenazine, chlorprothixene,fluphenazine, loxapine, mesoridazine, molindone, perphenazine, pimozide,piperacetazine, perchlorperazine, thioridazine, thiothixene,trifluoperazine, triflupromazine, pipamperone, amperozide, quietiapine,melperone, remoxipride, haloperidol, rispiridone, olanzepine,sertindole, ziprasidone, amisulpride, prochlorperazine, and thiothixene.

Anticonvulsants such as phenobarbital, phenytoin, primidone,carbamazepine, ethosuximide, lamotrigine, valproic acid, vigabatrin,felbamate, gabapentin, levetiracetam, oxcarbazepine, remacemide,tiagabine, and topiramate can also usefully be incorporated into thepharmaceutical compositions of the invention.

Muscle relaxants such as methocarbamol, orphenadrine, carisoprodol,meprobamate, chlorphenesin carbamate, diazepam, chlordiazepoxide andchlorzoxazone; anti-migraine agents such as sumitriptan, analeptics suchas caffeine, methylphenidate, amphetamine and modafinil; antihistaminessuch as chlorpheniramine, cyproheptadine, promethazine and pyrilamine,as well as corticosteroids such as methylprednisolone, betamethasone,hydrocortisone, prednisolone, cortisone, dexamethasone, prednisone,alclometasone, clobetasol, clocortrolone, desonide, desoximetasone,diflorasone, fluocinolone, fluocinonide, flurandrenolide, fluticasone,floromethalone, halcinonide, halobetasol, loteprednol, mometasone,prednicarbate, and triamcinolone can also be incorporated into thepharmaceutical compositions of the invention.

Ion channel blocking agents such as, for instance, the sodium ionchannel blocker, carbamazepine, as commonly used in the treatment oftinnitus, arrhythmia, ischemic stroke and epilepsy can beco-administered with or incorporated into the pharmaceuticalcompositions of the invention. Alternatively, or in addition, calciumion channel blockers, such as ziconotide, can also be used, as canantagonists of the ion channel associated with the NMDA receptor, suchas ketamine. There is evidence that at least some of these ion channelblockers can potentiate the analgesic effects of the kappa agonist andthereby reduce the dose required for affective pain relief. See forinstance, Wang et al., 2000, Pain 84: 271-81.

Suitable NSAIDs, or other non-opioid compounds with anti-inflammatoryand/or analgesic activity, that can be co-administered with orincorporated into the pharmaceutical compositions of the inventioninclude, but are not limited to one or more of the following:aminoarylcarboxylic acid derivatives such as etofenamate, meclofenamicacid, mefanamic acid, niflumic acid; arylacetic acid derivatives such asacemetacin, amfenac, cinmetacin, clopirac, diclofenac, fenclofenac,fenclorac, fenclozic acid, fentiazac, glucametacin, isoxepac, lonazolac,metiazinic acid, naproxin, oxametacine, proglumetacin, sulindac,tiaramide and tolmetin; arylbutyric acid derivatives such as butibufenand fenbufen; arylcarboxylic acids such as clidanac, ketorolac andtinoridine. arylpropionic acid derivatives such as bucloxic acid,carprofen, fenoprofen, flunoxaprofen, ibuprofen, ibuproxam, oxaprozin,phenylalkanoic acid derivatives such as flurbiprofen, piketoprofen,pirprofen, pranoprofen, protizinic acid and tiaprofenic acid;pyranocarboxylic acids such as etodolac; pyrazoles such as mepirizole;pyrazolones such as clofezone, feprazone, mofebutazone, oxyphinbutazone,phenylbutazone, phenyl pyrazolidininones, suxibuzone andthiazolinobutazone; salicylic acid derivatives such as aspirin,bromosaligenin, diflusinal, fendosal, glycol salicylate, mesalamine,1-naphthyl salicylate, magnesium salicylate, olsalazine andsalicylamide, salsalate, and sulfasalazine; thiazinecarboxamides such asdroxicam, isoxicam and piroxicam others such as ε-acetamidocaproic acid,acetaminophen, s-adenosylmethionine, 3-amino-4-hydroxybutyric acid,amixetrine, bendazac, bucolome, carbazones, cromolyn, difenpiramide,ditazol, hydroxychloroquine, indomethacin, ketoprofen and its activemetabolite 6-methoxy-2-naphthylacetic acid; guaiazulene, heterocylicaminoalkyl esters of mycophenolic acid and derivatives, nabumetone,nimesulide, orgotein, oxaceprol, oxazole derivatives, paranyline,pifoxime, 2-substituted-4,6-di-tertiary-butyl-s-hydroxy-1,3-pyrimidines,proquazone and tenidap, and cox-2 (cyclooxygenase II) inhibitors, suchas celecoxib or rofecoxib.

Suitable diuretics that can be co-administered with or incorporated intothe pharmaceutical preparations of the invention, include, for example,inhibitors of carbonic anhydrase, such as acetazolamide,dichlorphenamide, and methazolamide; osmotic diuretics, such asglycerin, isosorbide, mannitol, and urea; inhibitors of Na⁺—K⁺—2Cl⁻symport (loop diuretics or high-ceiling diuretics), such as furosemide,bumetanide, ethacrynic acid, torsemide, axosemide, piretanide, andtripamide; inhibitors of Na⁺—Cl⁻ symport (thiazide and thiazidelikediuretics), such as bendroflumethiazide, chlorothiazide,hydrochlorothiazide, hydroflumethazide, methyclothiazide, polythiazide,trichlormethiazide, chlorthalidone, indapamide, metolazone, andquinethazone; and, in addition, inhibitors of renal epithelial Na⁺channels, such as amiloride and triamterene, antagonists ofmineralocorticoid receptors (aldosterone antagonists), such asspironolactone, canrenone, potassium canrenoate, and eplerenone, which,together, are also classified as K⁺-sparing diuretics. One embodiment isco-formulation and/or co-administration of a loop or thiazide diuretictogether with a synthetic peptide amide of the invention for the purposeof a loop or thiazide diuretic dose-sparing effect, wherein the dose ofthe loop or thiazide diuretic is reduced to minimize undesired waterretention, and prevent or reduce hyponatremia, particularly in thecontext of congestive heart failure, as well as other medical conditionswhere decreasing body fluid retention and normalizing sodium balancecould be beneficial to a patient in need thereof. See R M Reynolds etal. Disorders of sodium balance Brit. Med. J. 2006;332:702-705.

The kappa opioid receptor-associated hyponatremia can be any disease orcondition where hyponatremia (low sodium condition) is present, e.g., inhumans, when the sodium concentration in the plasma falls below 135mmol/L, an abnormality that can occur in isolation or, more frequently,as a complication of other medical conditions, or as a consequence ofusing medications that can cause sodium depletion.

A further embodiment is co-formulation and/or co-administration of apotassium-sparing diuretic, e.g., a mineralocorticoid receptorantagonist, such as spironolactone or eplerenone, together with asynthetic peptide amide of the invention, for the purpose of enabling areduced dose of said potassium-sparing diuretic, wherein the dose ofsaid diuretic is reduced to minimize hyperkalemia or metabolic acidosis,e.g., in patients with hepatic cirrhosis.

In particular embodiments, the synthetic peptide amides of the inventionexhibit a long lasting duration of action when administered intherapeutically relevant doses in vivo. For instance, in someembodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 3 mg/kg of the synthetic peptideamide maintain at least about 50% of maximum efficacy in a kappa opioidreceptor-dependent assay at 3 hours post administration. In certainother embodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 0.1 mg/kg of the synthetic peptideamide maintain at least about 50% of maximum efficacy in a kappa opioidreceptor-dependent assay at 3 hours post administration. The maximumefficacy is operationally defined as the highest level of efficacydetermined for the particular kappa opioid receptor-dependent assay forall agonists tested.

In certain embodiments, the synthetic peptide amides of the inventionwhen administered to a mammal at a dose of 0.1 mg/kg maintain at leastabout 75% of maximum efficacy at 3 hours post administration. In stillother embodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 0.1 mg/kg maintain at least about90% of maximum efficacy at 3 hours post administration. In certain otherembodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 0.1 mg/kg maintain at least about95% of maximum efficacy at three hours post administration.

The invention further provides a method of treating or preventing akappa opioid receptor-associated disease or condition in a mammal,wherein the method includes administering to the mammal a compositioncontaining an effective amount of a synthetic peptide amide of theinvention. The mammal can be any mammal, such as a domesticated or feralmammal, or even a wild mammal. Alternatively, the mammal can be aprimate, an ungulate, a canine or a feline. For instance, and withoutlimitation, the mammal may be a pet or companion animal, such as ahigh-value mammal such as a thoroughbred or show animal; a farm animal,such as a cow, a goat, a sheep or pig; or a primate such as an ape ormonkey. In one particular aspect, the mammal is a human.

The effective amount can be determined according to routine methods byone of ordinary skill in the art. For instance, an effective amount canbe determined as a dosage unit sufficient to prevent or to treat a kappareceptor-associated disease or condition in the mammal. Alternatively,the effective amount may be determined as an amount sufficient toapproximate the EC₅₀ concentration or an amount sufficient toapproximate two or three times or up to about five or even about tentimes the EC₅₀ concentration in a therapeutically relevant body fluid ofthe mammal, for instance, where the body fluid is in direct appositionto a target tissue, such as the synovial fluid of an inflamed joint in apatient suffering from rheumatoid arthritis.

In one embodiment the synthetic peptide amide of the invention is apharmaceutical composition that includes an effective amount of thesynthetic peptide amide of the invention and a pharmaceuticallyacceptable excipient or carrier. In one aspect, the pharmaceuticalcomposition includes a synthetic peptide amide of the invention in anamount effective to treat or prevent a kappa opioid receptor-associatedcondition in a mammal, such as a human. In another aspect the kappaopioid receptor-associated condition is pain, inflammation, pruritis,edema, ileus, tussis or glaucoma.

In one embodiment the pharmaceutical composition of the inventionfurther includes one or more of the following compounds: an opioid, acannabinoid, an antidepressant, an anticonvulsant, a neuroleptic, acorticosteroid, an ion channel blocking agent or a non-steroidalanti-inflammatory drug (NSAID).

Pharmaceutical compositions of a synthetic peptide amide of theinvention and a pharmaceutically acceptable vehicle or carrier can beused to treat or prevent one or more of a variety of kappa opioidreceptor-associated diseases, disorders or conditions.

The kappa opioid receptor-associated disease, disorders or conditionpreventable or treatable with the synthetic peptide amides of theinvention can be any kappa opioid receptor-associated disease, disorderor condition, including but not limited to acute or chronic pain,inflammation, pruritis, hyponatremia, edema, ileus, tussis and glaucoma.For instance, the kappa opioid receptor-associated pain can beneuropathic pain, somatic pain, visceral pain or cutaneous pain. Somediseases, disorders, or conditions are associated with more than oneform of pain, e.g., post-operative pain (also known as post-surgicalpain) can have any or all of neuropathic, somatic, visceral, andcutaneous pain components, depending upon the type and extent ofsurgical procedure employed.

The kappa opioid receptor-associated inflammation can be anyinflammatory disease or condition including, but not limited tosinusitis, rheumatoid arthritis tenosynovitis, bursitis, tendonitis,lateral epicondylitis, adhesive capsulitis, osteomyelitis,osteoarthritic inflammation, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), ocular inflammation, otitic inflammation orautoimmune inflammation.

In another embodiment, the kappa opioid receptor-associated inflammatorydiseases and conditions preventable or treatable by the methods of thepresent invention is an inflammatory disease or condition characterizedby elevated levels of one or more proinflammatory cytokines, including,but not limited to tumor necrosis factor-α (TNF-α), interleukin 1β(IL-1β), interleukin 6 (IL-6) and matrix-metalloproteases (MMPS), suchas MMP-1 and MMP-3.

The invention further provides a method of prophylaxis or treatment of akappa opioid receptor-associated disease or condition in a mammal,wherein a synthetic peptide amide of the invention is co-administeredwith a reduced dose of a mu opioid agonist analgesic compound to producea therapeutic analgesic effect, the mu opioid agonist analgesic compoundhaving an associated side effect, (especially respiratory depression,sedation, euphoria, antidiuresis, nausea, vomiting, constipation, andphysical tolerance, dependence, and addiction). The reduced dose of themu opioid agonist analgesic compound (e.g. morphine or fentanyl or anyother opioid with substantial agonist activity at the mu opioidreceptor) administered by this method has lower associated side effectsthan the side effects associated with the dose of the compound necessaryto achieve the same therapeutic analgesic effect when administeredalone.

The invention also provides a method of treating or preventingperipheral hyperalgesia, wherein the method includes topically applyingor locally administering to a mammal in need of the treatment, aneffective amount of a composition that includes ananti-hyperalgesically-effective amount of a synthetic peptide amide ofthe invention in a vehicle formulated for topical application or localadministration.

The present invention also provides a method of treating, preventing orinhibiting a kappa opioid receptor-associated inflammatory disease orcondition. The inflammatory disease or condition can be any inflammatorydisease or condition such as for instance, cardiovascular inflammation,neurological inflammation, skeletal inflammation, muscular inflammation,gastrointestinal inflammation, ocular inflammation, otic inflammation,inflammation due to insect bites or inflammation due to wound healing.

The cardiovascular inflammatory disease or condition treatable orpreventable by the methods of the present invention can be anycardiovascular inflammatory disease or condition, such as cardiovascularinflammation due to a variety of causes, such as for instance,atherosclerosis, ischemia, restenosis or vasculitis.

Inflammatory diseases and conditions treatable or preventable by themethods of the present invention also include for example and withoutlimitation, asthma, Sjogren's syndrome, pulmonary inflammation,bronchitis, chronic airway inflammation and chronic obstructivepulmonary disease (COPD).

Other inflammatory diseases and conditions treatable or preventable bythe methods of the present invention also include immune diseases andconditions such as, without limitation, allergy, psoriasis, psoriaticarthritis, eczema, scleroderma, atopic dermatitis and systemic lupuserythematosus.

Still other inflammatory diseases and conditions treatable orpreventable by the methods of the present invention include arthritis,synovitis, osteomyelitis, rheumatoid arthritis, osteoarthritis andankylosing spondylitis. Furthermore, inflammatory diseases andconditions treatable or preventable by the methods of the invention alsoinclude septicemia and septic shock.

Inflammatory diseases and conditions treatable or preventable by themethods of the invention also include diabetes, glucose intolerance,insulin resistance and obesity.

Other inflammatory diseases and conditions treatable or preventable bythe methods of the present invention include colitis, ulcerativecolitis, Crohn's disease, IBD (inflammatory bowel disease) and IBS(Irritable bowel syndrome).

The present invention also provides methods of treatment or preventionof inflammatory diseases and conditions wherein the inflammatory diseaseor condition is due to tumor proliferation, tumor metastasis ortransplantation rejection.

The kappa opioid receptor-associated pruritis can be any pruriticdisease or condition such as, for instance, ocular pruritis, e.g.,associated with conjunctivitis, otitic pruritis, pruritis associatedwith end-stage renal disease, where many patients are receiving kidneydialysis, and other forms of cholestasis, including primary biliarycirrhosis, intrahepatic cholestasis of pregnancy, chronic cholestaticliver disease, uremia, malignant cholestasis, jaundice, as well asdermatological conditions such as eczema (dermatitis), including atopicor contact dermatitis, psoriasis, polycythemia vera, lichen planus,lichen simplex chronicus, pediculosis (lice), thyrotoxicosis, tineapedis, urticaria, scabies, vaginitis, anal pruritis associated withhemorrhoids and ,as well as insect bite pruritis and drug-inducedpruritis, such as mu opioid-induced pruritis.

The kappa opioid receptor-associated edema can be any edematous diseaseor condition such as, for instance, edema due to congestive heartdisease or to a syndrome of inappropriate antidiuretic hormone (ADH)secretion.

Kappa opioid receptor-associated ileus can be any ileus disease orcondition including, but not limited to, post-operative ileus andopioid-induced bowel dysfunction.

Kappa opioid receptor-associated neuropathic pain can be any neuropathicpain, such as, for instance, trigeminal neuralgia, diabetic pain, viralpain such as herpes zoster-associated pain, chemotherapy-induced pain,nerve-encroaching metastatic cancer pain, neuropathic pain associatedwith traumatic injury and surgical procedures, as well as variants ofheadache pain that are thought to have a neuropathic component, e.g.,migraine.

Kappa opioid-associated pain also includes ocular pain, such as thatfollowing photo-refractive keratectomy (PRK), ocular laceration, orbitalfloor fracture, chemical burns, corneal abrasion or irritation, or painassociated with conjunctivitis, corneal ulcers, scleritis, episcleritis,sclerokeratitis, herpes zoster ophthalmicus, interstitisal keratitis,acute iritis, keratoconjunctivitis sicca, orbital cellulites, orbitalpseudotumor, pemphigus, trachoma or uveitis.

Kappa opioid-associated pain also includes throat pain, particularlyassociated with inflammatory conditions, such as allergic rhinitis,acute bronchitis, the common cold, contact ulcers, herpes simplex virallesions, infectious mononucleosis, influenza, laryngeal cancer, acutelaryngitis, acute necrotizing ulcerative gingivitis, peritonsillarabscess, pharyngeal burns, pharyngitis, reflus laryngopharyngitis, acutesinusitis, and tonsillitis.

In addition, kappa opioid receptor-associated pain can be arthriticpain, kidney-stone, urinary tract stone, gallstone, and bile duct stonepain, dysmenorrhea, uterine cramping, endometriosis, mastitis,dyspepsia, post-surgical pain (such as, for instance, from appendectomy,open colorectal surgery, hernia repair, prostatectomy, colonicresection, gastrectomy, splenectomy, colectomy, colostomy, pelviclaparoscopy, tubal ligation, hysterectomy, vasectomy or cholecystecomy),post medical procedure pain (such as, for instance, after colonoscopy,cystoscopy, hysteroscopy or cervical or endometrial biopsy), otiticpain, breakthrough cancer pain, and pain associated with a GI disordersuch as IBD or IBS or other inflammatory conditions, particularly of theviscera (e.g., gastro-esophageal reflux disease, pancreatitis, acutepolynephritis, ulcerative colitis, acute pyelo-nephritis, cholecystitis,cirrhosis, hepatic abscess, hepatitis, duodenal or gastric ulcer,esophagitis, gastritis, gastroenteritis, colitis, diverticulitis,intestinal obstruction, ovarian cyst, pelvic inflammatory disease,perforated ulcer, peritonitis, prostatitis, interstitial cystitis), orexposure to toxic agents, such as insect toxins, or inflammation due tothe effects of drugs such as salicylates or NSAIDs.

The present invention provides a method of treating or preventing akappa opioid receptor-associated disease or condition in a mammal, suchas a human, wherein the method includes administering to the mammal acomposition comprising an effective amount of a synthetic peptide amideof the invention. In another embodiment the kappa opioidreceptor-associated condition is pain, inflammation (such as rheumatoidarthritic inflammation, osteoarthritic inflammation, IBD inflammation,IBS inflammation, ocular inflammation, otitic inflammation or autoimmuneinflammation), pruritis (such as atopic dermatitis,kidney-dialysis-associated pruritis, ocular pruritis, otitic pruritis,insect bite pruritis, or opioid-induced pruritis), edema, ileus, tussisor glaucoma. In one aspect, the pain is a neuropathic pain (such astrigeminal neuralgia, migraine, diabetic pain, viral pain,chemotherapy-induced pain or metastatic cancer pain), a somatic pain, avisceral pain or a cutaneous pain. In another aspect the pain isarthritic pain, kidney-stone pain, uterine cramping, dysmenorrhea,endometriosis, dyspepsia, post-surgical pain, post medical procedurepain, ocular pain, otitic pain, breakthrough cancer pain or painassociated with a GI disorder, such as IBD or IBS. In another aspect thepain is pain associated with surgery, wherein the surgery is pelviclaparoscopy, tubal ligation, hysterectomy and cholecystecomy.Alternatively, the pain can be pain associated with a medical procedure,such as for instance, colonoscopy, cystoscopy, hysteroscopy orendometrial biopsy. In a specific aspect, the atopic dermatitis can bepsoriasis, eczema or contact dermatitis. In another specific aspect, theileus is post-operative ileus or opioid-induced bowel dysfunction.

Kappa opioid receptor-associated pain includes hyperalgesia, which isbelieved to be caused by changes in the milieu of the peripheral sensoryterminal occur secondary to local tissue damage. Tissue damage (e.g.,abrasions, burns) and inflammation can produce significant increases inthe excitability of polymodal nociceptors (C fibers) and high thresholdmechanoreceptors (Handwerker et al. (1991) Proceeding of the VIth WorldCongress on Pain, Bond et al., eds., Elsevier Science Publishers B V,pp. 59-70; Schaible et al. (1993) Pain 55:5-54). This increasedexcitability and exaggerated responses of sensory afferents is believedto underlie hyperalgesia, where the pain response is the result of anexaggerated response to a stimulus. The importance of the hyperalgesicstate in the post-injury pain state has been repeatedly demonstrated andappears to account for a major proportion of thepost-injury/inflammatory pain state. See for example, Woold et al.(1993) Anesthesia and Analgesia 77:362-79; Dubner et al. (1994) In,Textbook of Pain, Melzack et al., eds., Churchill-Livingstone, London,pp. 225-242.

In another embodiment the kappa opioid receptor-associated condition ispain, inflammation (such as rheumatoid arthritic inflammation,osteoarthritic inflammation, IBD inflammation, IBS inflammation, ocularinflammation, otitic inflammation or autoimmune inflammation), pruritis(such as atopic dermatitis, kidney-dialysis-associated pruritis, ocularpruritis, otitic pruritis, insect bite pruritis, or opioid-inducedpruritis), edema, ileus, tussis or glaucoma. In one aspect, the pain isa neuropathic pain (such as trigeminal neuralgia, migraine, diabeticpain, viral pain, chemotherapy-induced pain or metastatic cancer pain),a somatic pain, a visceral pain or a cutaneous pain. In another aspectthe pain is arthritic pain, kidney-stone pain, uterine cramping,dysmenorrhea, endometriosis, dyspepsia, post-surgical pain, post medicalprocedure pain, ocular pain, otitic pain, breakthrough cancer pain orpain associated with a GI disorder, such as IBD or IBS. In anotheraspect the pain is pain associated with surgery, wherein the surgery ispelvic laparoscopy, tubal ligation, hysterectomy and cholecystecomy.Alternatively, the pain can be pain associated with a medical procedure,such as for instance, colonoscopy, cystoscopy, hysteroscopy orendometrial biopsy. In a specific aspect, the atopic dermatitis can bepsoriasis, eczema or contact dermatitis. In another specific aspect, theileus is post-operative ileus or opioid-induced bowel dysfunction.

In another embodiment the kappa opioid receptor-associated condition isa kappa opioid receptor-associated condition preventable or treatable bysodium and potassium-sparing diuresis, also known as aquaresis. Anexample of such kappa opioid receptor-associated conditions preventableor treatable by administering a synthetic peptide amide of the inventionincludes edema. The edema may be due to any of a variety of diseases orconditions, such as congestive heart disease or syndrome ofinappropriate ADH secretion.

In another embodiment the kappa opioid receptor-associated condition ishyponatremia or other edematous disease. The kappa opioidreceptor-associated hyponatremia or edema can be any hyponatremic oredematous disease or condition such as, for instance, hyponatremia andedema associated with congestive heart failure or to a syndrome ofinappropriate antidiuretic hormone (ADH) secretion, or hyponatremia thatis associated with intensive diuretic therapy with thiazides and/or loopdiuretics. The synthetic peptide amides of the invention exhibit asignificant sodium-sparing and potassium-sparing aquaretic effect, whichis beneficial in the treatment of edema-forming pathological conditionsassociated with hyponatremia and/or hypokalemia. Accordingly, thesynthetic peptide amides of the invention also have utility in methodsof treating or preventing hyponatremia-related conditions, examples ofwhich are provided below. Hyponatremia-related conditions can becategorized according to volume status as hypervolemic, euvolemic, orhypovolemic.

Hypervolemic hyponatremia is usually caused by an increase in total bodywater level as may be observed in cases of congestive heart failure,nephrotic syndrome and hepatic cirrhosis.

Euvolemic hyponatremia is often found in the syndrome of inappropriateantidiuretic hormone (ADH) secretion and may also be associated withpneumonia, small-cell lung cancer, polydipsia, cases of head injury, andorganic causes (e.g., use of certain drugs, such as haloperidol) or apsychogenic cause.

Hypovolemic hyponatremia is due to a relative decrease in total bodysodium level and may be associated with, for instance and withoutlimitation, diuretic use, cases of interstitial nephritis or excessivesweating.

These forms of hyponatremia can be further classified according to theconcentration of sodium in the urine (i.e., whether the concentration isgreater than or less than thirty millimoles per liter. See: R M Reynoldset al. Disorders of sodium balance, Brit. Med. J. 2006;332:702-705.

The kappa opioid receptor-associated hyponatremia can be any disease orcondition where hyponatremia (low sodium condition) is present, e.g., inhumans, when the sodium concentration in the plasma falls below 135mmol/L, an abnormality that can occur in isolation or, more frequently,as a complication of other medical conditions, or as a consequence ofusing medications that can cause sodium depletion.

In addition to these conditions, numerous other conditions areassociated with hyponatremia including, without limitation: neoplasticcauses of excess ADH secretion, including carcinomas of lung, duodenum,pancreas, ovary, bladder, and ureter, thymoma, mesothelioma, bronchialadenoma, carcinoid, gangliocytoma and Ewing's sarcoma; infections suchas: pneumonia (bacterial or viral), abscesses (lung or brain),cavitation (aspergillosis), tuberculosis (lung or brain), meningitis(bacterial or viral), encephalitis and AIDS; vascular causes such as:cerebrovascular occlusions or hemorrhage and cavernous sinus thrombosis;neurologic causes such as: Guillan-Barre syndrome, multiple sclerosis,delirium tremens, amyotrophic lateral sclerosis, hydrocephalus,psychosis, peripheral neuropathy, head trauma (closed and penetrating),CNS tumors or infections and CNS insults affecting hypothalamicosmoreceptors; congenital malformations including: agenesis of corpuscallosum, cleftlip/palate and other midline defects; metabolic causessuch as: acute intermittent porphyria, asthma, pneurothorax andpositive-pressure respiration; drugs such as: thiazide diuretics,acetaminophen, barbiturates, cholinergic agents, estrogen, oralhypoglycemic agents, vasopressin or desmopressin, high-dose oxytocin,chlorpropamide, vincristine, carbamezepine, nicotine, phenothiazines,cyclophosphamide, tricyclic antidepressants, monoamine oxidaseinhibitors and serotonin reuptake inhibitors; administration of excesshypotonic fluids, e.g., during hospitalization, surgery, or during orafter athletic events (i.e., exercise-associated hyponatremia), as wellas use of low-sodium nutritional supplements in elderly individuals. Seefor example, Harrison's Principles of Internal Medicine, 16th Ed.(2005), p. 2102.

Other conditions associated with hyponatremia incude renal failure,nephrotic syndrome (membranous nephropathy and minimal change disease),cachexia, malnutrition, rhabdomyolysis, surgical procedures, electivecardiac catheterization, blood loss, as well as hypercalcemia,hypokalemia, and hyperglycemia with consequent glycosuria leading toosmotic diuresis.

The invention also provides a method of treating or preventing aneurodegenerative disease or condition in a mammal, such as a human,wherein the method includes administering to the mammal a compositionthat includes an effective amount of a synthetic peptide amide asdescribed above. The neurodegenerative disease or condition can be anyneurodegenerative disease or condition, such as for instance, ischemia,anoxia, stroke, brain injury, spinal cord injury or reperfusion injury.Alternatively, the neurodegenerative disease or condition can be aneurodegenerative disease of the eye. Particular neurodegenerativediseases of the eye treatable or preventable by the method of theinvention include glaucoma, macular degeneration, retinal ischemicdisease and diabetic neuropathy.

In certain embodiments the invention provides methods of prevention ortreatment of certain neuronal diseases and conditions, such as diseasesand conditions having a neurodegenerative component. Synthetic peptideamides of the invention can be administered in an amount effective toprotect neuronal cells against the effects of pathology or injury thatwould lead to neurodegeneration and/or neuronal cell death of theuntreated cells. For example, several diseases or conditions of the eyethat have a neurodegenerative component can be prevented or treated byadministration of an effective amount of the synthetic peptide amides ofthe invention. Such diseases and conditions of the eye include glaucoma,macular degeneration, retinal ischemic disease and diabetic neuropathy.Progression of these diseases and conditions is believed to involveneurodegeneration or neuronal cell death, for example by programmed celldeath (apoptosis) in which the neuronal cells are committed to a pathwaythat without intervention would lead to cell death. It has been foundthat development or progression of these diseases and conditions can beprevented, or at least slowed, by treatment with kappa opioid receptoragonists. This improved outcome is believed to be due to neuroprotectionby the kappa opioid receptor agonists. See for instance, Kaushik et al.“Neuroprotection in Glaucoma” (2003) J. Postgraduate Medicine vol. 49(1): pp. 90-95.

In the case of glaucoma it is believed that prophylaxis and treatment byadministration of kappa opioid receptor agonists is mediated by at leasttwo distinct activities induced by activation of the kappa opioidreceptor: neuroprotection and reduction of intraocular pressure (IOP).While not wishing to be bound by theory, it is believed thatneuroprotection is due, at least in part, to induction of atrialnatriuretic peptide (ANP) in the eye, leading to protection againstoxidative damage and other insults.

Abnormally high intraocular pressure is also believed to be a factorleading to the development of glaucoma. Elevated intraocular pressurecan also be prevented or treated by administration of kappa opioidreceptor agonists by three separate activities triggered by activationof the receptor: reduction in secretion of aqueous humor, increasedoutflow of aqueous humor and aquaresis (sodium- and potassium-sparingdiuresis, resulting in loss of water).

The invention also provides a method of treating or preventing akappa-receptor-associated disease or condition of the eye of a mammal,such as high intraocular pressure (IOP). The method includesadministering to the mammal a composition that includes an effectiveamount of a synthetic peptide amide as described above. In one aspect ofthe invention, the synthetic peptide amide is administered topically. Inanother aspect, the synthetic peptide amide is administered as animplant.

In other embodiments the invention provides methods of prevention ortreatment of certain cardiovascular diseases and conditions having acellular degenerative component. Synthetic peptide amides of theinvention can be administered in an amount effective to protectmyocardial cells against the effects of pathology or injury that wouldlead to degeneration and/or cell death of the untreated cells. Forexample, several cardiovascular diseases or conditions can be preventedor treated by administration of an effective amount of the syntheticpeptide amides of the invention. Such cardiovascular diseases andconditions include, without limitation, coronary heart disease,ischemia, cardiac infarct, reperfusion injury and arrhythmia. See forexample, Wu et al. “Cardioprotection of Preconditioning by MetabolicInhibition in the Rat Ventricular Myocyte—Involvement of kappa OpioidReceptor” (1999) Circulation Res vol. 84: pp. 1388-1395. See also Yu etal. “Anti-Arrythmic Effect of kappa Opioid Receptor Stimulation in thePerfused Rat Heart: Involvement of a cAMP-Dependent Pathway” (1999) JMol Cell Cardiol. vol. 31(10): pp. 1809-1819.

Diseases and conditions of other tissues and organs that can beprevented or treated by administration of an effective amount of thesynthetic peptide amides of the invention include, but are not limitedto ischemia, anoxia, stroke, brain or spinal cord injury and reperfusioninjury.

Another form of kappa opioid receptor-associated pain treatable orpreventable with the synthetic peptide amides of the invention ishyperalgesia. In one embodiment, the method includes administering aneffective amount of a synthetic peptide amide of the invention to amammal suffering from or at risk of developing hyperalgesia to prevent,ameliorate or completely alleviate the hyperalgesia.

The synthetic peptide amides of the invention can be administered bymethods disclosed herein for the treatment or prevention of anyhyperalgesic condition, such as, but without limitation, a hyperalgesiccondition associated with allergic dermatitis, contact dermatitis, skinulcers, inflammation, rashes, fungal irritation and hyperalgesicconditions associated with infectious agents, burns, abrasions, bruises,contusions, frostbite, rashes, acne, insect bites/stings, skin ulcers,mucositis, gingivitis, bronchitis, laryngitis, sore throat, shingles,fungal irritation, fever blisters, boils, Plantar's warts, surgicalprocedures or vaginal lesions. For instance, the synthetic peptideamides of the invention can be administered topically to a mucosalsurface, such as the mouth, esophagus or larynx, or to the bronchial ornasal passages. Alternatively, the synthetic peptide amides of theinvention can be administered topically to the vagina or rectum/anus.

Moreover, the synthetic peptide amides of the invention can beadministered by methods disclosed herein for the treatment or preventionof any hyperalgesic condition associated with burns, abrasions, bruises,abrasions (such as corneal abrasions), contusions, frostbite, rashes,acne, insect bites/stings, skin ulcers (for instance, diabetic ulcers ora decubitus ulcers), mucositis, inflammation, gingivitis, bronchitis,laryngitis, sore throat, shingles, fungal irritation (such as athlete'sfoot or jock itch), fever blisters, boils, Plantar's warts or vaginallesions (such as vaginal lesions associated with mycosis or sexuallytransmitted diseases). Methods contemplated for administration of thesynthetic peptide amides of the invention for the treatment orprevention of hyperalgesia include those wherein the compound istopically applied to a surface in the eyes, mouth, larynx, esophagus,bronchial, nasal passages, vagina or rectum/anus.

Hyperalgesic conditions associated with post-surgery recovery can alsobe addressed by administration of the synthetic peptide amides of theinvention. The hyperalgesic conditions associated with post-surgeryrecovery can be any hyperalgesic conditions associated with post-surgeryrecovery, such as for instance, radial keratectomy, tooth extraction,lumpectomy, episiotomy, laparoscopy and arthroscopy.

Hyperalgesic conditions associated with inflammation are alsoaddressable by administration of the synthetic peptide amides of theinvention. Periodontal inflammation, orthodontic inflammation,inflammatory conjunctivitis, hemorrhoids and venereal inflammations canbe treated or prevented by topical or local administration of thesynthetic peptide amides of the invention.

The invention also provides a method of inducing diuresis in a mammal inneed thereof. The method includes administering to the mammal acomposition comprising an effective amount of a synthetic peptide amideof the invention as described above.

The invention further provides a method of inducing prolactin secretionin a mammal. The method includes administering to the mammal acomposition comprising an effective amount of a synthetic peptide amideof the invention as described above. The method of inducing prolactinsecretion is suitable for treating a mammal, such as a human sufferingfrom insufficient lactation, inadequate lactation, sub-optimallactation, reduced sperm motility, an age-related disorder, type Idiabetes, insomnia or inadequate REM sleep. In a particular aspect, themethod includes co-administering the synthetic peptide amide with areduced dose of a mu opioid agonist analgesic compound to produce atherapeutic analgesic effect, the compound having an associated sideeffect, wherein the reduced dose of the compound has a lower associatedside effect than the side effect associated with the dose of the muopioid agonist analgesic compound necessary to achieve the therapeuticanalgesic effect when administered alone.

The present invention also provides a method of binding a kappa opioidreceptor in a mammal, the method includes the step of administering tothe mammal a composition containing an effective amount of a syntheticpeptide amide of the present invention. The effective amount can bedetermined according to routine methods by one of ordinary skill in theart. For instance, the effective amount can be determined as a dosageunit sufficient to bind kappa opioid receptors in a mammal and cause anantinociceptive effect, an anti-inflammatory effect, an aquareticeffect, or an elevation of serum prolactin levels or any other kappaopioid receptor-responsive effect. Alternatively, the effective amountmay be determined as an amount sufficient to approximate the EC₅₀ in abody fluid of the mammal, or an amount sufficient to approximate two orthree, or up to about five or even about ten times the EC₅₀ in atherapeutically relevant body fluid of the mammal.

EXAMPLES General Experimental Synthetic Methods:

Amino acid derivatives and resins were purchased from commercialproviders (Novabiochem, Bachem, Peptide International and PepTechCorporation). Other chemicals and solvents were purchased fromSigma-Aldrich, Fisher Scientific and VWR. The compounds herein weresynthesized by standard methods in solid phase peptide chemistryutilizing both Fmoc and Boc methodology. Unless otherwise specified, allreactions were performed at room temperature.

The following standard references provide guidance on generalexperimental setup, and the availability of required starting materialand reagents: Kates, S. A., Albericio, F., Eds., Solid Phase Synthesis,A Practical Guide, Marcel Dekker, New York, Basel, (2000); Bodanszky,M., Bodanszky, A., Eds., The Practice of Peptide Synthesis, SecondEdition, Springer-Verlag, (1994); Atherton, E., Sheppard, R. C., Eds.,Solid Phase Peptide Synthesis, A Practical Approach, IRL Press at OxfordUniversity Press, (1989); Stewart, J. M., Young, J. D., Solid PhaseSynthesis, Pierce Chemical Company, (1984); Bisello, et al., J. Biol.Chem. 273, 22498-22505 (1998); and Merrifield, R. B., J. Am. Chem. Soc.85, 2149-2154 (1963).

Abbreviations used herein: ACN: acetonitrile; Aloc: allyloxy-carbonyl;Boc: tert-butoxycarbonyl; BOP:benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate; Cbz: Benzyloxycarbonyl; Cbz-OSu:Nα-(Benzyloxy-carbonyloxy) succinimide;DBU:1,8-diazabicyclo[5.4.0]undec-7-ene; DCM: Dichloro-methane; Dde:1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; DIC:N,N′-diiso-propylcarbodiimide; DIEA: N,N-diisopropylethylamine; DMF:N,N-dimethyl-formamide; Fmoc: 9-fluorenylmethoxycarbonyl; HATU:2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate; HBTU:2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate; HOBt: 1-hydroxybenzotriazole; HPLC: highperformance liquid chromatography; i: iso; ivDde:1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl; NMM:4-methyl morpholino; NMP: N-methylpyrrolidinone; All: allyl; o-NBS-Cl:o-nitrobenzenesulfonyl chloride; Pbf:2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PyB OP:benzotriazole-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophosphate;RP: reversed phase; TBTU:2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate;TEAP: triethylammonium phosphate; TFA: trifluoroacetic acid; TIS:triisopropylsilane; TMOF: trimethyl orthoformate; TMSOTf: trimethylsilyltrifluoromethanesulfonate; Trt: trityl.

Peptides synthesized by Fmoc methodology were cleaved with a mixture ofTFA/TIS/H₂O (v/v/v=95:2.5:2.5). The cleavage step in the Boc methodologywas accomplished either with a mixture of HF/anisole (v/v=9:1) or with amixture of TMSOTf/TFA/m-cresol (v/v/v=2:7:1).

Coupling reactions in peptide chain elongation were carried out eithermanually or on a peptide synthesizer and mediated by coupling reagentswith a 2 to 4-fold excess amino acid derivatives. The coupling reagentsused in the synthesis of the various compounds of the invention werechosen from the following combinations: DIC/HOBt, HATU/DIEA, HBTU/DIEA,TBTU/DIEA, PyB OP/DIEA, and BOP/DIEA.

Deprotection of the side chain of amino acid in position No. 4(designated Xaa₄ in the final synthetic peptide amide product) of resinbound peptides was achieved as follows: Peptides were assembled startingfrom Xaa₄ and progressively adding Xaa₃, then Xaa₂ and finally, Xaa₁.The side chain protecting groups of the diamino acid introduced at Xaa₄were selectively removed as follows: (i) N-Dde or N-ivDde groups wereremoved by 2-4% hydrazine in DMF. See Chabra, S. R., et al., TetrahedronLett. 39:1603-1606 (1998) and Rohwedder, B., et al., Tetrahedron Lett.,39: 1175 (1998); (ii) N-Aloc: removed by 3 eq. (Ph₃P)₄Pd inCHCl₃/AcOH/NMM (v/v/v=37:2:1). See Kates, S. A., et al. in “PeptidesChemistry, Structure and Biology, Proc. 13^(th) American PeptideSymposium”, Hodges, R. S. and Smith, J. A. (Eds), ESCOM, Leiden, 113-115(1994).

When peptides were assembled with Boc protection methodology, the sidechain protecting group of the diamino acids introduced at Xaa₄ wasN-Fmoc, which was removed by 20-30% piperidine in DMF.

Isopropylation of the terminal nitrogen on the side chain of amino acidat Xaa₄ of resin bound peptides was achieved as follows: Afterdeprotection, the resin bound peptide with the free ω-amino function atXaa₄ was reacted with a mixture of acetone and NaBH(OAc)₃ in TMOFproducing the resin bound N-ω-isopropyl peptide.

Monomethylation of the terminal nitrogen on the side chain of amino acidat Xaa₄ of resin bound peptides: To synthesize resin bound N-ω-methylpeptides, the free ω-amino function was first derivatized witho-nitrobenzene-sulfonyl chloride (o-NBS-Cl; Biron, E.; Chatterjee, J.;Kessler, H. Optimized selective N-methylation of peptides on solidsupport. J. Pep. Sci. 12:213-219 (2006). The resulting sulfonamide wasthen methylated with a mixture of dimethylsulphate and1,8-diaza-bicyclo[5.4.0]undec-7-ene in NMP. The o-NBS protecting groupwas subsequently removed by a mixture of mercaptoethanol and1,8-diazabicyclo[5.4.0]undec-7-ene in NMP.

Guanylation of the terminal nitrogen on the side chain of amino acid atXaa₄ of resin bound peptides: After deprotection, the resin boundpeptide with the free ω-amino function in position No. 4 was reactedwith a mixture of 1H-pyrazole-1-carboxamidine hydrochloride(Bernatowicz, M. S., et al., J. Org. Chem. 57, 2497-2502 (1992) and DIEAin DMF producing the resin bound N-ω-guanidino peptide.

Peptides were purified by preparative HPLC in triethylammonium phosphate(TEAP) or trifluoroacetic acid (TFA) buffers. When required, thecompounds were finally converted to trifluoroacetate or acetate saltsusing conventional HPLC methodology. Fractions with purity exceeding 97%were pooled and lyophilized. Purity of the synthesized peptides wasdetermined by analytical RP-HPLC.

Analytical RP-HPLC was performed on a Waters 600 multisolvent deliverysystem with a Waters 486 tunable absorbance UV detector and a Waters 746data module. HPLC analyses of peptides were carried out using a VydacC₁₈ column (0.46×25 cm, 5 μm particle size, 300 Å pore size) at a flowrate of 2.0 ml/min Solvents A and B were 0.1% TFA in H₂O and 0.1% TFA in80% ACN/20% H₂O, respectively. Retention times (t_(R)) are given inminutes. Preparative RP-HPLC was accomplished using a Vydac C₁₈preparative cartridge (4.7×30 cm, 15-20 μm particle size, 300 Å poresize) at a flow rate of 100 ml/min, on a Waters Prep LC 2000 preparativechromatograph system with a Waters 486 tunable absorbance UV detectorand a Servogor 120 strip chart recorder. Buffers A and B were 0.1% TFAin H₂O and 0.1% TFA in 60% ACN/40% H₂O, respectively. HPLC analysis ofthe final compound was performed on a Hewlett Packard 1090 LiquidChromatograph using a Phenomenex Synergi MAX-RP C₁₂ column (2.0×150 mm,4 μm particle size, 80 Å pore size) at a flow rate of 0.3 ml/min at 40°C. Buffers A and B were 0.01% TFA in H₂O and 0.01% TFA in 70% ACN/30%H₂O, respectively. The identity of the synthetic peptide amides wasconfirmed by electrospray mass spectrometry. Mass spectra were recordedon a Finnigan LCQ mass spectrometer with an ESI source.

Synthetic Peptide Amides (1)-(103):

Example 1 Synthesis of compound (1)D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[4-amidinohomopiperazine amide]

The amino acid derivatives used were Cbz-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, and Fmoc-D-Lys(Dde)-OH. The fully protected resin boundpeptide was synthesized manually starting from p-nitro-phenylcarbonateWang resin (5.0 g, 4.4 mmol; Novabiochem). The attachment ofhomopiperazine to the resin was achieved by mixing it with a solution ofhomopiperazine (8.7 g, 87 mmol; Acros Organics) in DCM (100 mL)overnight at room temperature. The resin was washed with DMF and DCM anddried in vacuo. The resulting homopiperazine carbamate Wang resin (5.1g; homopiperazine-[carbamate Wang resin]) was split into severalportions and a portion of 1.5 g (1.3 mmol) was used to continue thepeptide synthesis. DIC/HOBt mediated single couplings were performedwith a 3-fold excess of amino acid derivatives. The Fmoc group wasremoved with 25% piperidine in DMF. Upon completion of peptide chainelongation, the resin was treated with 4% hydrazine in DMF for 3×3 minfor Dde removal. The resin was washed with DMF and DCM and dried invacuo. The resulting peptide resin (2.4 g;Cbz-D-Phe-D-Phe-DLeu-DLys-homopiperazine-[carbamate Wang resin]) wassplit again and a portion of 0.6 g (0.3 mmol) was used for subsequentderivatization (N-methylation).

Methylation of the ω-amino function of D-Lys at Xaa₄ was carried out inthree steps: (i) [o-NBS Protection]: The resin-bound peptide (0.3 mmol)was first treated with a solution o-NBS-Cl (0.4 g, 2 mmol) and collidine(0.7 ml, 5 mmol) in NMP (7 ml) at room temperature for 30 minutes. Theresin was then washed with NMP. (ii) [N-Methylation]: The resin-boundo-NBS protected peptide was then reacted with a solution of1,8-diazabicyclo[5.4.0]undec-7-ene (0.5 ml, 3 mmol) and dimethylsulfate(1.0 ml, 10 mmol; Aldrich) in NMP (7 ml) at room temperature for 5minutes. The resin was then washed with NMP and the washing process wasrepeated once. (iii) [o-NBS Deprotection]: The peptide resin was treatedwith a solution of mercaptoethanol (0.7 ml, 10 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (0.8 ml, 5 mmol) in NMP (7 ml) atroom temperature for 5 minutes. The resin was then washed with NMP andthe washing process was repeated once.

To protect the resulting N-methyl secondary amine of D-Lys at Xaa₄, theresin-bound methylated peptide was reacted with a solution of Cbz-OSu (6mmol) in DMF (7 ml). The resin was washed with DMF and DCM and dried invacuo. The peptide was then cleaved from the resin by treatment with asolution of TFA/DCM (15 ml, v/v=1:1) at room temperature for 2 hours.The resin was then filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (0.3 mmol;Cbz-D-Phe-D-Phe-D-Leu-D-Lys(Me,Cbz)-[homopiperazine amide]) wasprecipitated from diethyl ether.

For guanylation of the homopiperazine at the C-terminus, the abovepeptide (0.3 mmol) was treated with a solution of1H-Pyrazole-1-carboxamidine hydrochloride (0.4 g, 3.0 mmol) and DIEA(0.5 ml, 6 mmol) in DMF (3 ml) overnight at room temperature. Aceticacid and H₂O were added to quench the reaction and the solution wasfrozen and dried on a lyophilizer to give the desired protected peptide,Cbz-D-Phe-D-Phe-D-Leu-D-Lys(Me,Z)-[4-Amidinohomopiperazine amide] (0.6g).

For final deprotection/hydrolysis, the above peptide (0.6 g) was treatedwith a mixture of TMSOTf/TFA/m-cresol (10 ml, v/v/v=2:7:1) at roomtemperature for two hours. The mixture was evaporated and the crudesynthetic peptide amide (0.6 g) was precipitated from diethyl ether.

For purification, the above-derived crude synthetic peptide amide (0.6g) was dissolved in 0.1% TFA in H₂O (50 ml) and the solution was loadedonto an HPLC column and purified using TFA buffer system (buffers A=0.1%TFA in H₂O and B=0.1% TFA in 60% ACN/40% H₂O). The compound was elutedwith a linear gradient of buffer B, 25% B to 75% B over 30 min,t_(R)=37% B. The fractions with purity exceeding 97% were pooled,frozen, and dried on a lyophilizer to give the purified syntheticpeptide amide as white amorphous powder (153 mg). HPLC analysis:t_(R)=14.41 min, purity 99.8%, gradient 5% B to 25% B over 20 min; MS(MH⁺): expected molecular ion mass 692.5, observed 692.5.

Example 2 Synthesis of compound (2):D-Phe-D-Phe-D-Leu-D-Lys-[ω(4-aminopiperidine-4-carboxylic acid)]—OH

See Biron et al., Optimized selective N-methylation of peptides on solidsupport. J. Peptide Science 12: 213-219 (2006). The amino acidderivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Fmoc-D-Lys(Dde)-OH, and N-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylicacid. HPLC and MS analyses were performed as described in the synthesisof compound (1) described above.

The fully protected resin-bound peptide was synthesized manuallystarting from 2-Chlorotrityl chloride resin (1.8 g, 0.9 mmol; PeptideInternational). Attachment ofN-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylic acid followed by peptidechain elongation and deprotection of Dde in D-Lys(Dde) at Xaa₄ wascarried out according to the procedure described in the synthesis ofcompound (1). See above. The resulting peptide resin (0.9 mmol;Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)-[2-Cl-Trt resin]) was split and a portion of 0.3 mmol was used forsubsequent cleavage. The peptide resin (0.3 mmol) was then treated witha mixture of TFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperaturefor 90 minutes. The resin was then filtered and washed with TFA. Thefiltrate was evaporated in vacuo and the crude synthetic peptide amide(0.3 mmol; D-Phe-D-Phe-D-Leu-D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]—OH) was precipitated from diethyl ether.

For purification, the crude synthetic peptide amide (0.3 mmol) wasdissolved in 2% acetic acid in H₂O (50 ml) and the solution was loadedonto an HPLC column and purified using TEAP buffer system with a pH 5.2(buffers A=TEAP 5.2 and B=20% TEAP 5.2 in 80% ACN). The compound waseluted with a linear gradient of buffer B, 7% B to 37% B over 60minutes. Fractions with purity exceeding 95% were pooled and theresulting solution was diluted with two volumes of water. The dilutedsolution was then loaded onto an HPLC column for salt exchange andfurther purification with a TFA buffer system (buffers A=0.1% TFA in H₂Oand B=0.1% TFA in 80% ACN/20% H₂O) and a linear gradient of buffer B, 2%B to 75% B over 25 minutes. Fractions with purity exceeding 97% werepooled, frozen, and dried on a lyophilizer to yield the purifiedsynthetic peptide amide as white amorphous powder (93 mg). HPLCanalysis: t_(R)=16.43 min, purity 99.2%, gradient 5% B to 25% B over 20min; MS (MH⁺): expected molecular ion mass 680.4, observed 680.3.

Compound (2) was also prepared using the following amino acidderivatives: Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, andBoc-4-amino-1-Fmoc-(piperidine)-4-carboxylic acid.

The fully protected resin-bound peptide was synthesized manuallystarting from 2-Chlorotrityl chloride resin (PS 1% DVB, 500 g, 1 meq/g).The resin was treated withBoc-4-amino-1-Fmoc-4-(piperidine)-4-carboxylic acid (280 g, 600 mmol) ina mixture of DMF, DCM and DIEA (260 mL of each) was added. The mixturewas stirred for 4 hours and then the resin was capped for 1 h by theaddition of MeOH (258 mL) and DIEA (258 mL).

The resin was isolated and washed with DMF (3×3 L). The resin containingthe first amino acid was treated with piperidine in DMF (3×3 L of 35%),washed with DMF (9×3 L) and Fmoc-D-Lys(Boc)-OH (472 g) was coupled usingPyBOP (519 g) in the presence of HOBt (153 g) and DIEA (516 mL) and inDCM/DMF (500 mL/500 mL) with stiffing for 2.25 hours. The dipeptidecontaining resin was isolated and washed with DMF (3×3.6 L). The Fmocgroup was removed by treatment with piperidine in DMF

(3×3.6 L of 35%) and the resin was washed with DMF (9×3.6 L) and treatedwith Fmoc-D-Leu-OH (354 g), DIC (157 mL) and HOBt (154 g) in DCM/DMF(500 mL/500 mL) and stirred for 1 hour. Subsequent washing with DMF(3×4.1 L) followed by cleavage of the Fmoc group with piperidine in DMF(3×4.2 L of 35%) and then washing of the resin with DMF (9×4.2 L)provided the resin bound tripeptide. This material was treated withFmoc-D-Phe-OH (387 g), DIC (157 mL) and HOBt (153 g) in DCM/DMF (500mL/500 mL) and stirred overnight. The resin was isolated, washed withDMF (3×4.7 L) and then treated with piperidine in DMF (3×4.7 L of 35%)to cleave the Fmoc group and then washed again with DMF (9×4.7 L). Thetetrapeptide loaded resin was treated with Fmoc-D-Phe-OH (389 g), DIC(157 mL) and HOBt (154 g) in DCM/DMF (500 mL/500 mL) and stirred for2.25 hours. The resin was isolated, washed with DMF (3×5.2 L) and thentreated piperidine (3×5.2 L of 35%) in DMF. The resin was isolated, andwashed sequentially with DMF (9×5.2 L) then DCM (5×5.2 L). It was driedto provide a 90.4% yield of protected peptide bound to the resin. Thepeptide was cleaved from the resin using TFA/water (4.5 L, 95/5), whichalso served to remove the Boc protecting groups. The mixture wasfiltered, concentrated (⅓) and then precipitated by addition to MTBE (42L). The solid was collected by filtration and dried under reducedpressure to give crude synthetic peptide amide.

For purification, the crude synthetic peptide amide was dissolved in0.1% TFA in H₂O and purified by preparative reverse phase HPLC (C18)using 0.1% TFA/water—ACN gradient as mobile phase. Fractions with purityexceeding 95% were pooled, concentrated and lyophilized to provide puresynthetic peptide amide (>95.5% pure). Ion exchange was conducted usinga Dowex ion exchange resin, eluting with water. The aqueous phase wasfiltered (0.22 μm filter capsule) and freeze-dried to give the acetatesalt of the synthetic peptide amide (2) with overall yield, 71.3%, >99%purity.

Hydrochloride, hydrobromide and fumarate counterions were evaluated fortheir ability to form crystalline salts of synthetic peptide amide (2).Approximately 1 or 2 equivalents (depending on desired stoichiometry) ofhydrochloric acid, hydrobromic acid or fumaric acid, as a dilutesolution in methanol (0.2-0.3 g) was added to synthetic peptide amide(2) (50-70 mg) dissolved in methanol (0.2-0.3 g). Each individual saltsolution was added to isopropyl acetate (3-5 mL) and the resultingamorphous precipitate was collected by filtration and dried at ambienttemperature and pressure. Crystallization experiments were carried outby dissolving the 10-20 mg of the specific amorphous salt obtained abovein 70:30 ethanol-water mixture (0.1-0.2 g) followed by the addition ofethanol to adjust the ratio to 90:10 (˜0.6-0.8 mL). Each solution wasthen seeded with solid particles of the respective precipitated salt.Each sample tube was equipped with a magnetic stir bar and the samplewas gently stirred at ambient temperature. The samples were periodicallyexamined by plane-polarized light microscopy. Under these conditions,the mono- and di-hydrochloride salts, the di-hydrobromide salt and themono-fumarate salt crystallized as needles of 20 to 50 μm in length witha thickness of about 1 μm.

Example 3 Synthesis of compound (3):D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]—OH

The synthesis was started with 0.3 mmol of the peptide resin:Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)-[2-Cl-Trt resin], which was prepared during the synthesis ofcompound (2) as described below. HPLC and MS analyses were alsoperformed as described in the synthesis of compound (2) above.

For the methylation of the w-amino function of D-Lys at Xaa₄, athree-step procedure as described in the synthesis of compound (1) wasfollowed. See description above. The resin-bound methylated peptide(Boc-D-Phe-D-Phe-D-Leu-(e-Me)D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)42-Cl-Trt resin]) was then treated with a mixture of TFA/TIS/H₂O(15 ml, v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resinwas then filtered and washed with TFA. The filtrate was evaporated invacuo and the crude synthetic peptide amide (0.3 mmol;D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[ω(4-amino-piperidine-4-carboxylicacid)]—OH) was precipitated from diethyl ether.

The crude synthetic peptide amide (0.3 mmol) was purified by preparativeHPLC according to the protocol described in the synthesis of compound(2). See above. Fractions with purity exceeding 97% were pooled, frozen,and dried on a lyophilizer to yield the purified synthetic peptide amideas white amorphous powder (185 mg). HPLC analysis: t_(R)=16.93 min,purity 99.2%, gradient 5% B to 25% B over 20 min; MS (MH⁺): expectedmolecular ion mass 694.4, observed 694.4.

Example 4 Synthesis of compound (4):D-Phe-D-Phe-D-Leu-D-Lys-[N-(4-piperidinyl)-L-proline]—OH

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, Fmoc-D-Lys(Dde)-OH, andN-(1-Fmoc-piperidin-4-yl)-L-proline. HPLC and MS analyses were performedas described in the synthesis of compound (1). See detailed descriptionabove. Couplings were mediated by HATU/DIEA.

The fully protected resin-bound peptide was synthesized manuallystarting from 2-Chlorotrityl chloride resin (3.2 g, 2.4 mmol; NeoMPS).The attachment of the first amino acid to the resin was achieved bytreatment with a mixture of N-(1-Fmoc-piperidin-4-yl)-L-proline (2.0 g,4.8 mmol) and DIEA (3.3 ml, 19 2 mmol) in DCM (40 ml) and DMF (10 ml) atroom temperature for 4 hours. The resin was washed with 3× DCM/MeOH/DIEA(v/v/v=17:2:1) and 3× DCM and dried in vacuo. The resulting resin (3.7g; N-(4-piperidinyl)-L-proline-[2-Cl-Trt resin]) was split into severalportions and a portion of 1.9 g (1.2 mmol) was used to continue thepeptide synthesis. HATU/DIEA-mediated single couplings were performedwith a 3-fold excess of amino acid derivatives. The Fmoc group wasremoved with 25% piperidine in DMF. Upon completion of peptide chainelongation, the resin was treated with 4% hydrazine in DMF three timesfor 3 min each to remove Dde. The resin was washed with DMF and DCM anddried in vacuo. The resulting peptide resin (2.1 g;Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin]) was split again and a portion of 0.7 g (0.4 mmol) was used forsubsequent cleavage. The peptide resin was treated with a mixture ofTFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperature for 90minutes. The resin was filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (220 mg,D-Phe-D-Phe-D-Leu-D-Lys4N-(4-piperidinyl)-L-proline]—OH) wasprecipitated from diethyl ether.

For purification, the above crude peptide (220 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 25 min, t_(R)=43% B. Fractions withpurity exceeding 97% were pooled, frozen, and dried on a lyophilizer togive the purified synthetic peptide amide as white amorphous powder (89mg). HPLC analysis: t_(R)=18.22 min, purity 99.5%, gradient 5% B to 25%B over 20 min; MS (MH⁺): expected molecular ion mass 734.5, observed734.4.

Example 5 Synthesis of compound (5):D-Phe-D-Phe-D-Leu-D-Har-[N-(4-piperidinyl)-L-proline]—OH

The peptide-resin:Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin], which was prepared during the synthesis of compound (4)described above, was used as the starting material. HPLC and MS analyseswere performed as described in the synthesis of compound (1) above.

For guanylation of the ω-amino function of D-Lys at Xaa₄, the peptideresin (0.7 g, 0.4 mmol) was treated with a mixture of1H-Pyrazole-1-carboxamidine hydrochloride (0.6 g, 4.0 mmol) and DIEA(0.7 ml, 4.0 mmol) in DMF (15 ml) overnight at room temperature. Theresin was washed with DMF and DCM and dried in vacuo. The peptide wasthen cleaved from the resin by treatment with a mixture of TFA/TIS/H₂O(15 ml, v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resinwas then filtered and washed with TFA. The filtrate was evaporated invacuo and the crude synthetic peptide amide (170 mg;D-Phe-D-Phe-D-Leu-D-Har-N-(4-piperidinyl)-L-proline]—OH) wasprecipitated from diethyl ether.

For purification, the above crude synthetic peptide amide (170 mg) wasdissolved in 0.1% TFA in H₂O (50 ml) and the solution was loaded onto anHPLC column and purified using a TFA buffer system (buffers A=0.1% TFAin H₂O and B=0.1% TFA in 60% ACN/40% H₂O). The compound was eluted witha linear gradient of buffer B, 25% B to 75% B over 25 min, t_(R)=46% B.Fractions with purity exceeding 97% were pooled, frozen, and lyophilizedto yield the purified synthetic peptide amide as white amorphous powder(81 mg). HPLC analysis: t_(R)=19.42 min, purity 100%, gradient 5% B to25% B over 20 min; MS (MH⁺): expected molecular ion mass 776.5, observed776.5.

Example 6 Synthesis of compound (6):D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[N-(4-piperidinyl)-L-proline]—OH

Synthesis was initiated with 0.7 g (0.4 mmol) of the peptide resin,Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin], which was prepared during the synthesis of compound (4) asdescribed above. HPLC and MS analyses were performed as described in thesynthesis of compound (1) above. In this case, the Xaa₁-Xaa₄peptide waspre-synthesized and coupled.

For the methylation of the ω-amino function of D-Lys at Xaa₄, athree-step procedure was followed as described in the synthesis ofcompound (1) above. The resin-bound methylated peptide(Boc-D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin]) was then treated with a mixture of TFA/TIS/H₂O (15 ml,v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resin wasfiltered and washed with TFA. The filtrate was evaporated in vacuo andcrude peptide (200 mg;D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[N-(4-piperidinyl)-L-proline]OH)precipitated from diethyl ether.

For purification, the above crude peptide (200 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution loaded onto an HPLC column andpurified using a TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof 25% to 75% buffer B, over 30 min, t_(R)=42% B. Fractions with purityexceeding 97% were pooled, frozen, and dried on a lyophilizer to yieldthe purified synthetic peptide amide as white amorphous powder (41 mg).HPLC analysis: t_(R)=18.66 min, purity 98.1%, gradient 5% B to 25% Bover 20 min; MS (MH⁺): expected molecular ion mass 748.5, observed748.5.

Example 7 Synthesis of (7): D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazineamide]

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, and Fmoc-D-Arg(Pbf)-OH. HPLC and MS analyses wereperformed as in the synthesis of compound (1) described above. The fullyprotected resin bound peptide was synthesized on a SYMPHONY MultipleSynthesizer (Protein Technology Inc.) starting from the homopiperazinecarbamate Wang resin (0.35 mmol; homopiperazine-[carbamate Wang resin])that was prepared during the synthesis of compound (1). HBTU/DIEAmediated single couplings with a 4-fold excess of amino acid derivativeswere performed. The Fmoc group was removed with 25% piperidine in DMF.Upon completion of the automated synthesis, the peptide resin(Boc-D-Phe-D-Phe-D-Leu-D-Arg(Pbf)-[homopiperazine amide]) wastransferred into a manual peptide synthesis vessel and treated with amixture of TFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperature for90 minutes. The resin was filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (380 mg;D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide]) was precipitated fromdiethyl ether.

For purification, the above crude peptide (380 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using a TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 25 min, t_(R)=36% B. Fractions withpurity exceeding 97% were pooled, frozen, and lyophilized to give thepurified synthetic peptide amide as white amorphous powder (222 mg).HPLC analysis: t_(R)=16.75 min, purity 100%, gradient 2% B to 22% B over20 min; MS (MH⁺): expected molecular ion mass 664.4, observed 664.5.

Example 8 Synthesis of compound (8):D-Phe-D-Phe-D-Leu-D-Har-[ω(4-aminopiperidine-4-carboxylic acid]—OH

This compound was prepared essentially according to the proceduredescribed above for the synthesis of compound (5) except thatN-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylic acid was substituted forN-(1-Fmoc-piperidin-4-yl)-L-proline in the attachment to 2-Cl-Trt resin.Final purified synthetic peptide amide: amorphous powder, 85 mg in yieldin a synthesis scale of 1 mmol HPLC analysis: t_(R)=17.87 min, purity100%, gradient 5% B to 25% B over 20 min; MS (MH⁺): expected molecularion mass 722.4, observed 722.5.

Example 9 Synthesis of compound (9):D-Phe-D-Phe-D-Leu-(ε-iPr)D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]—OH

Synthesis was initiated from 0.15 mmol of the peptide resin,Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)-[2-Cl-Trt resin]), which was prepared during the synthesis ofcompound (2) above. For isopropylation of the ω-amino function of D-Lysat Xaa₄, the peptide resin was treated with a mixture of sodiumtriacetoxyborohydride (3 mmol) and acetone (6 mmol) in TMOF (10 mL) for4 h at room temperature. Subsequent cleavage and purification steps werecarried out according to the procedure described in the synthesis ofcompound (2). Final purified synthetic peptide amide: amorphous powder,67 mg in yield. HPLC analysis: t_(R)=19.29 min, purity 98.4%, gradient5% B to 25% B over 20 min; MS (MH⁺): expected molecular ion mass 722.5,observed 722.5.

Example 10 Synthesis of compound (10):D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[ω(4-aminopiperidine-4-carboxylicacid)]—OH

The amino acid derivatives used were Boc-D-Phe-OH, Boc-D-Phe-OH,Boc-D-Leu-OH, Boc-D-Dap(Fmoc)-OH, andN-Fmoc-amino-(4-N-Boc-piperidinyl)carboxylic acid. HPLC and MS analyseswere performed as described in the synthesis of compound (1). The fullyprotected resin-bound peptide was synthesized manually starting with4-Fmoc-hydrazinobenzoyl AM NovaGel resin (3 mmol; Novabiochem). The Fmocprotecting group on the starting resin was first removed by 25%piperidine in DMF and the resin was then treated with a mixture ofN-Fmoc-amino-(4-N-Boc-piperidinyl)carboxylic acid (7.5 mmol), PyBOP (7.5mmol), and DIEA (15 mmol) in DMF overnight at room temperature. The Fmocgroup on the attached amino acid was replaced by o-NBS in two steps: (i)Fmoc removal by 25% piperidine in DMF. (ii) o-NBS protection accordingthe procedure described in the synthesis of compound (1). The resultingpeptide resin, N-o-NBS-amino-(4-N-Boc-piperidinyl)carboxylicacid4hydrazinobenzoyl AM NovaGel resin], was split into several portionsand a portion of 1 mmol was used to continue the peptide synthesis.PyBOP/DIEA mediated single couplings were performed with a 3-fold excessof amino acid derivatives. The Boc group was removed with 30% TFA inDCM. Upon completion of peptide chain elongation, the resin was treatedwith 2% DBU in DMF for 2×8 min for Fmoc removal, followed by guanylationof the ω-amino function of D-Dap at Xaa₄. The final o-NBS deprotectionwas carried out according to the procedure described in the synthesis ofcompound (1).

For oxidative cleavage, the dried peptide resin was mixed with a mixtureof Cu(OAc)₂ (1 mmol), pyridine (4 mmol), and DBU (2 mmol) in 5% H₂O inDMF and let air bubble through the resin for 6 h at room temperature.The resin was filtered and washed with DMF and the filtrated wasevaporated in vacuo. The residue,Boc-D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[ω(4-aminopiperidine-4-carboxylicacid)]—OH, was treated with 95% TFA in H₂O for Boc removal. The solutionwas evaporated in vacuo and the crude peptide (1 mmol;D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[ω(4-aminopiperidine-4-carboxylicacid)]—OH) was precipitated from diethyl ether.

Purification of the above crude peptide was achieved according to theprotocol described in the synthesis of compound (2). The purifiedsynthetic peptide amide was an amorphous powder (16 mg). HPLC analysis:t_(R)=16.97 min, purity 99.9%, gradient 5% B to 25% B over 20 min; MS(MH⁺): expected mass 680.4, observed 680.4.

Examples 11-23 Synthesis of Compounds (11)-(23)

These compounds were prepared according to the procedure described inthe synthesis of compound (10), except that appropriate Boc-amino acidderivatives were substituted for Boc-D-Dap(Fmoc)-OH in the coupling atXaa₄. See U.S. Pat. No. 7,713,937.

Example 24 Synthesis of compound (24):D-Phe-D-Phe-D-Leu-D-Arg-[4-Amidinohomopiperazine amide]

The compound was prepared by guanylation of the homopiperazine atC-terminus of Cbz-D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide], whichwas synthesized according to the procedure described in the synthesis ofcompound (7), described above. Subsequent cleavage and purification werecarried out according to the procedure described in the synthesis ofcompound (1), above. Final purified peptide: amorphous powder, 102 mg inyield in a synthesis scale of 0.3 mmol HPLC analysis: t_(R)=17.34 min,purity 98.4%, gradient 2% B to 22% B over 20 min; MS (MH⁺): expectedmolecular ion mass 706.5, observed 706.5.

Example 25 Synthesis of compound (25):D-Phe-D-Phe-D-Leu-D-Lys-[2,8-diazaspiro[4,5]decan-1-one amide]

To a suspension of Boc-D-Phe-OH intermediate I-1 (7.96 g, 30.0 mmol),D-Leu-OBn p-TsOH intermediate 1-2 (11.80 g, 30.0 mmol), HOBt monohydrate(4.46 g, 33.0 mmol) and DIEA (8.53 g, 66.0 mmol) in anhydrous THF (250mL) cooled in an ice-water bath was added EDCI (6.33 g, 33.0 mmol) infour portions over 20 minutes with 5 minutes between each addition. Thesuspension was stirred overnight from a starting temperature of 0° C. toroom temperature. After evaporation of THF, the residue was dissolved inethyl acetate and washed sequentially with 10% citric acid, saturatedNaHCO₃ and water. The organic phase was dried over sodium sulfate andevaporated under reduced pressure. The residue was dissolved in DCM,passed through a silica gel plug and eluted with 20% ethyl acetate inhexanes. Eluant was evaporated to give the pure Boc-D-Phe-D-Leu-OBn,intermediate 1-3 (12.40 g, 88%) as a clear oil. LC-MS: m/z=469 (MH).

Intermediate I-3 (12.40 g, 26.5 mmol) was dissolved in DCM (50 mL). TFA(25 mL) was added and the solution was stirred at room temperature for 2hours. After evaporation of DCM and TFA, the residue was azeotroped withtoluene twice to give the TFA salt of D-Phe-Leu-OBn, intermediate 1-4.This crude dipeptide was suspended in THF, to which Boc-D-Phe-OH (6.36g, 24 mmol), HOBt monohydrate (4.04 g, 26.4 mmol) and DIEA (8.7 mL, 50.0mmol) was added at 0° C. EDCI (6.33 g, 6 4 mmol) was added in fourportions over 20 minutes with 5 minutes between each addition. Thesuspension was stirred from 0° C. to room temperature overnight. Afterevaporation of THF, the residue was dissolved in ethyl acetate andwashed sequentially with 10% citric acid, saturated NaHCO₃ and water.The organic phase was dried over sodium sulfate and evaporated underreduced pressure. The residue was recrystalized from 400 mLacetone/hexanes (1:3) to give 9.1 g pure product. The mother liquor wasevaporated and again recrystalized from acetone/hexanes (1:3) to give2.0 g product. The total yield was 11.1 g (68% for two steps). LC-MS:m/z=616 (MH).

In a flask flushed with nitrogen was added wet palladium on carbon (1.8g) and a solution of Boc-D-Phe-D-Phe-D-Leu-OBn, intermediate I-5 (11.1g, 18.05 mmol) in methanol (50 mL). The mixture was stirred under ahydrogen balloon overnight. After filtration through celite, methanolwas evaporated under reduced pressure. The residue was dissolved inacetone (20 mL) and slowly added to 500 mL water with 25 mL of 1N HClunder vigorous stirring. Pure product Boc-D-Phe-D-Phe-D-Leu-OH,intermediate I-6 was obtained by filtration 9.4 g (99%). LC-MS: m/z=526(MH).

To a solution of intermediate I-6 (2.06 g, 3.90 mmol), D-Lys(Boc)-OAllhydrochloride (1.26 g, 3.90 mmol) and DIEA (1.7 ml, 9.8 mmol) in DMF wasadded TBTU (1.56 g, 4.88 mmol) in three portions over 15 min at 0° C.After stirring overnight from a starting temperature of 0° C. to roomtemperature, DMF was evaporated under high vacuum. The crude reactionmixture was precipitate in 400 ml ice water and filtered to collect theprecipitate, Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OAll intermediate I-7(2.60 g), which was used without further purification for the next step.

To a solution of intermediate I-7 (2.60 g, 3.3 mmol) in MeCN (75 mL) wasadded pyrrolidine (1.1 ml, 13.3 mmol) and palladiumtetrakis(triphenylphosphine) (400 mg, 0.35 mmol) at 0° C. The reactionmixture was stirred at room temperature for 3 hours and evaporated todryness. Residue was purified by reverse phase column chromatographywith 30% MeCN/water to 90% MeCN/water to give pure acid, intermediateI-8 (2.0 g, 80%) after evaporation of acetonitrile/water. LC-MS: m/z=754(MH⁺).

To a solution of the acid, intermediate I-8 (150 mg, 0.20 mmol), theamine HNR_(a)R_(b), 2,8-diazaspiro[4,5]decan-1-one (57 mg, 0.30 mmol)and DIEA (175 ul, 1.0 mmol) in DMF (5 mL) was added HBTU (11.3 mg, 0.3mmol) at 0° C. After stiffing overnight from a starting temperature of0° C. to room temperature, DMF was evaporated under reduced pressure.The residue was stirred with 4N HCl in 1,4-dioxane (2.0 mL) at roomtemperature for 1 hour. After removal of dioxane, the residue wasdissolved in water and purified by reverse phase column chromatographywith a gradient of 10% MeCN/water to 60% MeCN/water in 30 minutes togive pure synthetic peptide amide, compound (25) (108 mg, 78% yield forthe two steps) after evaporation of solvent. LC-MS: m/z=690 (MH⁺).

Examples 26-37 Synthesis of Compounds (26)-(37)

These compounds were prepared according to the procedure described inthe synthesis of compound (26) above except for the substitution of theappropriate amine used in the final step as described in detail in U.S.Pat. No. 7,713,937.

Example 38 Synthesis of compound (38):D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]—OH

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, andFmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine dihydrochloride. Thefully protected resin-bound peptide was synthesized on a SYMPHONYMultiple Synthesizer (Protein Technology Inc.) starting from2-Chlorotrityl chloride resin (0.4 mmol; Novabiochem). The attachment ofthe first amino acid to the resin was achieved by treatment with amixture of Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazinedihydrochloride (0.24 g, 0.5 mmol; Chem-Impex International Inc.) andDIEA (0.35 mL, 2 mmol) in DMF (7 ml) at room temperature for 4 hours.The resin was washed with 3× DCM/MeOH/DIEA (v/v/v=17:2:1) and 3× DCM.The subsequent peptide chain elongation was achieved by HBTU/DIEAmediated single couplings with a 3-fold excess of amino acidderivatives. The Fmoc group was removed by 25% piperidine in DMF. Forcleavage, the final peptide resin was treated with a mixture ofTFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperature for 90minutes. The resin was filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (0.2 g,D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]—OH)was precipitated from diethyl ether.

For purification, the above crude peptide (0.2 g) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using a TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 30 min, t_(R)=35% B. The fractions withpurity exceeding 97% were pooled, frozen, and dried on a lyophilizer toyield the purified synthetic peptide amide as a white amorphous powder(101 mg). HPLC analysis: t_(R)=16.24, purity 100%, gradient 5% B to 25%B over 20 min; MS (MH⁺): expected molecular ion mass 709.4, observed709.4.

Examples 39-48 Synthesis of Compounds (39)-(48)

These compounds were prepared according to the procedure described inthe synthesis of compound (38) using the appropriate amino acidderivatives as detailed in U.S. Pat. No. 7,713,937.

Example 49 Synthesis of compound (49):D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]—NH₂

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, andFmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine dihydrochloride. Thefully protected resin-bound peptide was synthesized on a SYMPHONYMultiple Synthesizer (Protein Technology Inc.) starting from Rink AmideAM resin (0.3 mmol; Novabiochem). The attachment of the first amino acidto the resin was achieved by treatment with a mixture ofFmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazine dihydrochloride (0.48g, 1 mmol; Chem-Impex International Inc.), HBTU (0.38 g, 1 mmol) andDIEA (0.53 mL, 3 mmol) in DMF (7 ml) at room temperature for 3 hours.The resin was washed with three times in DMF.

Subsequent peptide chain elongation was achieved by HBTU/DIEA mediatedsingle couplings with a 3-fold excess of amino acid derivatives. TheFmoc group was removed by 25% piperidine in DMF. For cleavage, the finalpeptide resin was treated with a mixture of TFA/TIS/H₂O (15 ml,v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resin wasfiltered and washed with TFA. The filtrate was evaporated in vacuo andthe crude synthetic peptide amide (0.1 g,D-Phe-D-Phe-D-Leu-D-Orn-[4-(2-aminoethyl)-1-carboxymethyl-piperazine]—NH₂)was precipitated from diethyl ether.

For purification, the above crude synthetic peptide amide (0.1 g) wasdissolved in 0.1% TFA in H₂O (50 ml) and the solution was loaded onto anHPLC column and purified using a TFA buffer system (buffers A=0.1% TFAin H₂O and B=0.1% TFA in 60% ACN/40% H₂O). The compound was eluted witha linear gradient of buffer B, 25% B to 75% B over 30 min, t_(R)=38% B.The fractions with purity exceeding 97% were pooled, frozen, and driedon a lyophilizer to yield the purified synthetic peptide amide as awhite amorphous powder (36 mg). HPLC analysis: t_(R)=16.59, purity99.5%, gradient 2% B to 22% B over 20 min; MS (MH⁺): expected mass708.5, observed 708.3.

Examples 50 and 51 Synthesis of Compounds (50) and (51)

These compounds were prepared according to the procedure described inthe synthesis of compound (49) For compounds (50) and (51)N-(1-Fmoc-piperidin-4-yl)-L-proline (NeoMPS) andFmoc-4-amino-1-carboxymethyl-piperidine (NeoMPS) were respectivelysubstituted for Fmoc-4-(2-aminoethyl)-1-carboxymethyl-piperazinedihydrochloride in the attachment to Rink Amide AM resin.

Example 52 Synthesis of compound (52):D-Phe-D-Phe-D-Leu-D-Orn-[4-(N-methyl)amidinohomopiperazine amide]

Compound (52) was prepared by guanylation of the homopiperazine atC-terminus of Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine amide],which was synthesized according to the procedure described below.

The guanylation reagent S-Methyl-N-methylisothiourea hydroiodide wasprepared by reacting 1,3-dimethyl-2-thiourea with methyl iodide inanhydrous methanol. See McKay, A. F.; Hatton, W. G. Synthesis of CyclicGuanidino Acids. J. Am. Chem. Soc. (1955), 78, 1618-1620 and Kennedy, K.J., et al. A Facile Route to Cyclic and Acyclic Alkyl-Arginines.Synthetic Communications, (1998), 28, 741-746.

For synthesis of Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazineamide], the amino acid derivatives used were Z-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, and Fmoc-D-Orn(Cbz)-OH. The fully protected resin boundpeptide was synthesized manually starting from p-nitrophenylcarbonateWang resin (5.0 g, 4.4 mmol; Novabiochem). The attachment ofhomopiperazine to the resin was achieved by mixing a solution ofhomopiperazine (8.7 g, 87 mmol; Acros Organics) in DCM (100 ml)overnight at room temperature. The resin was washed with 3× DMF and 3×DCM. The subsequent peptide chain elongation was achieved byHBTU/DIEA-mediated single couplings with a 3-fold excess of amino acidderivatives. The Fmoc group was removed by 25% piperidine in DMF. Forcleavage, the final synthetic peptide amide resin was treated with amixture of TFA/DCM (100 mL, v/v=1:1) at room temperature for 2 hours.The resin was filtered and washed with DCM. The filtrate was evaporatedin vacuo and the residue was dissolved in 0.1% TFA in 60% ACN/40% H₂O.The solution was frozen, and dried on a lyophilizer to give the crudepeptide intermediate Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-homopiperazine(4.4 g). For purification, the crude peptide (4.4 g) was divided intotwo portions and each portion was dissolved in 0.1% TFA in 30% ACN (100ml). Each solution was loaded onto an HPLC column and purified using aTFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1% TFA in 60%ACN/40% H₂O). The compound was eluted with a linear gradient of bufferB, 40% B to 100% B over 25 min, t_(R)=87% B. The fractions with purityexceeding 97% were pooled, frozen, and dried on a lyophilizer to yieldthe purified peptide intermediate as a white amorphous powder (3.0 g).

For guanylation of the homopiperazine at C-terminus, the above peptideintermediate (210 mg, 0.3 mmol) was treated with a mixture ofS-Methyl-N-methylisothiourea hydroiodide (1.4 g, 6 mmol) and DIEA (1.0mL, 12 mmol) in DMF (4 mL) at room temperature for 18 days. The mixturewas evaporated in vacuo and the residue was dissolved in 0.1% TFA in 30%ACN/70% H₂O and the solution was loaded onto an HPLC column and purifiedusing a TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1% TFA in60% ACN/40% H₂O). The compound was eluted with a linear gradient ofbuffer B, 70% B to 100% B over 30 min, t_(R)=85% B. The fractions withpurity exceeding 97% were pooled, frozen, and dried on a lyophilizer toyield the purified peptide intermediate,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[N-methylhomopiperazine-1-carboximidamideamide], as white amorphous powder (100 mg).

For final deprotection/hydrolysis, the above purified peptide (100 mg)was treated with a mixture of TMSOTf/TFA/m-cresol (10 ml, v/v/v=2:7:1)at room temperature for 2 hours. The mixture was evaporated in vacuo andthe crude peptide (100 mg) was precipitated from diethyl ether.

For purification, the above crude peptide (100 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 25 min, t_(R)=43% B. The fractions withpurity exceeding 97% were pooled, frozen, and dried on a lyophilizer toyield the purified synthetic peptide amide as a white amorphous powder(53 mg). HPLC analysis: t_(R)=17.99 min, purity 99.4%, gradient 2% B to22% B over 20 min; MS (MH⁺): expected molecular ion mass 678.4, observed678.5.

Example 53 Synthesis of compound (53):D-Phe-D-Phe-D-Leu-D-Orn-[4-amidinohomopiperazine amide]

Synthesis was initiated from 0.2 mmol of the peptide intermediate,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine amide], which wasprepared during the synthesis of compound (52). For guanylation of thehomopiperazine at C-terminus, the peptide was treated with a solution of1H-Pyrazole-1-carboxamidine hydrochloride (0.4 g, 3.0 mmol) and DIEA(0.5 ml, 6 mmol) in DMF (3 ml) overnight at room temperature. Aceticacid and H₂O were added to quench the reaction and the solution wasfrozen and dried on a lyophilizer to give the desired protected peptide,Cbz-DPhe-DPhe-DLeu-DOm(Cbz)-[4-Amidinohomopiperazine amide] (0.2 mmol).The subsequent deprotection/hydrolysis and HPLC purification werecarried out according to the procedure described in the synthesis ofcompound (52). Final purified synthetic peptide amide: amorphous powder,74 mg in yield. HPLC analysis: t_(R)=10.10 min, purity 98.7%, gradient10% B to 30% B over 20 min; MS (MH⁺): expected mass 664.4, observed664.5.

Example 54 Synthesis of compound (54):D-Phe-D-Phe-D-Leu-D-Orn-[4-(4,5-dihydro-1H-imidazol-2-yl)homopiperazineamide]

Synthesis was initiated from 0.2 mmol of the peptide intermediate,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine amide], which wasprepared during the synthesis of compound (52). For guanylation of thehomopiperazine at C-terminus, the peptide was treated with a solution of2-Methylthio-2-imidazoline hydroiodide (730 mg, 3.0 mmol; Aldrich) andDIEA (0.5 ml, 6 mmol) in DMF (3 ml) for four days at room temperature.Acetic acid and H₂O were added to quench the reaction and the solutionwas frozen and dried on a lyophilizer to give the desired protectedpeptide,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[4-(4,5-dihydro-1H-imidazol-2-yl)homopiperazineamide] (0.2 mmol). The subsequent deprotection/hydrolysis and HPLCpurification were carried out according to the procedure described inthe synthesis of compound (52). The final purified synthetic peptideamide was an amorphous powder, 46 mg in yield. HPLC analysis:t_(R)=10.89 min, purity 100%, gradient 10% B to 30% B over 20 min; MS(MH⁺): expected molecular ion mass 690.4, observed 690.5.

Example 55 Synthesis of compound (55):D-Phe-D-Phe-D-Leu-D-Orn-[4-ethylhomopiperazine amide]

Synthesis was initiated from 0.3 mmol of the peptide intermediate,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine amide], which wasprepared during the synthesis of compound (52). For ethylation of thehomopiperazine at C-terminus, the peptide was treated with a solution ofiodoethane (0.4 mmol; Aldrich) and DIEA (0.5 ml, 6 mmol) in DMF (3 ml)for 1 day at room temperature. The subsequent deprotection/hydrolysisand HPLC purification were carried out according to the proceduredescribed in the synthesis of compound (52). Final purified syntheticpeptide amide: amorphous powder, 75 mg in yield. HPLC analysis:t_(R)=10.43 min, purity 98.4%, gradient 10% B to 30% B over 20 min; MS(MH⁺): expected mass 650.4, observed 650.3.

Example 56 Synthesis of compound (56):D-Phe-D-Phe-D-Leu-D-Orn-[homopiperazine amide]

Synthesis was initiated from 0.3 mmol of the peptide intermediate,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Cbz)-[homopiperazine amide], which wasprepared during the synthesis of compound (52). The peptide washydrolyzed with a mixture of TMSOTf/TFA/m-cresol (10 ml, v/v/v=2:7:1)and the crude product was purified by preparative HPLC according to theprocedure described in the synthesis of compound (52). Final purifiedsynthetic peptide amide: amorphous powder, 225 mg in yield. HPLCanalysis: t_(R)=16.43 min, purity 100%, gradient 2% B to 22% B over 20min; MS (MH⁺): expected molecular ion mass 622.4, observed 622.4.

Example 57 Synthesis of compound (57):D-Phe-D-Phe-D-Leu-(δ-Me)D-Orn-[4-amidinohomopiperazine amide]

The amino acid derivatives used were Z-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, and Fmoc-D-Orn(Aloc)-OH. The fully protected resin boundpeptide was synthesized manually starting from p-nitrophenyl-carbonateWang resin (5.0 g, 4.4 mmol; Novabiochem). The attachment ofhomo-piperazine to the resin was achieved by mixing it with a solutionof homopiperazine (8.7 g, 87 mmol; Acros Organics) in DCM (100 mL)overnight at room temperature. The resin was washed with DMF and DCM anddried in vacuo. The resulting homopiperazine carbamate Wang resin (5.1g; homopiperazine-[carbamate Wang resin]) was split into severalportions and a portion of 1.1 g (1 mmol) was used to continue thepeptide synthesis. DIC/HOBt mediated single couplings were performedwith a 3-fold excess of amino acid derivatives. The Fmoc group wasremoved with 25% piperidine in DMF. Upon completion of peptide chainelongation, the resin was treated with Pd(PPh₃)₄ (3.5 g, 3.0 mmol;Aldrich) in a mixture of CHCl₃/AcOH/NMM (60 ml, v/v/v=37:2:1) underArgon atmosphere at room temperature for 3 h for Aloc removal. The resinwas washed with DMF and DCM and dried in vacuo. The resulting peptideresin (1.8 g; Z-D-Phe-D-Phe-D-Leu-D-Orn-homopiperazine-[carbamate Wangresin]) was split again and a portion of 0.9 g (0.5 mmol) was used forsubsequent derivatization (N-methylation).

Methylation of the ω-amino function of D-Orn at Xaa₄ was carried out inthree steps: (i) [o-NBS Protection]: The resin-bound peptide (0.5 mmol)was first treated with a solution o-NBS-Cl (0.4 g, 2 mmol) and collidine(0.7 ml, 5 mmol) in NMP (7 ml) at room temperature for 30 minutes. Theresin was then washed with NMP. (ii) [N-Methylation]: The resin-boundo-NBS protected peptide was then reacted with a solution of1,8-diazabicyclo[5.4.0]undec-7-ene (0.5 ml, 3 mmol) and dimethylsulfate(1.0 ml, 10 mmol; Aldrich) in NMP (7 ml) at room temperature for fiveminutes. The resin was then washed with NMP and the N-methylationprocess was repeated once. (iii) [o-NBS Deprotection]: The peptide resinwas treated with a solution of mercaptoethanol (0.7 ml, 10 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (0.8 ml, 5 mmol) in NMP (7 ml) atroom temperature for five minutes. The resin was then washed with NMPand the deprotection process was repeated once.

To protect the resulting N-methyl secondary amine of D-Orn at Xaa4, theresin-bound methylated peptide was reacted with a solution of Cbz-OSu (6mmol) in DMF (7 ml). The resin was washed with DMF and DCM and dried invacuo. The peptide was then cleaved from the resin by treatment with asolution of TFA/DCM (15 ml, v/v=1:1) at room temperature for 2 hours.The resin was then filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (0.5 mmol;Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Me,Cbz)-[homopiperazine amide]) was obtainedas an oil.

For guanylation of the homopiperazine at the C-terminus, a portion ofthe above peptide (0.3 mmol) was treated with a solution of1H-Pyrazole-1-carboxamidine hydrochloride (0.4 g, 3.0 mmol) and DIEA(0.5 ml, 6 mmol) in DMF (3 ml) overnight at room temperature. Aceticacid and H₂O were added to quench the reaction and the solution wasfrozen and dried on a lyophilizer to give the desired protected peptide,Z-D-Phe-D-Phe-D-Leu-D-Orn(Me,Z)-[4-Amidinohomopiperazine amide] (0.3mmol).

For final deprotection/hydrolysis, the above peptide (0.3 mmol) wastreated with a mixture of TMSOTf/TFA/m-cresol (10 ml, v/v/v=2:7:1) atroom temperature for 2 hours. The mixture was evaporated affording thecrude peptide (0.3 mmol) as an oil. The preparative HPLC purification ofthe crude peptide was carried out according to the procedure asdescribed in the synthesis of compound (38). Final purified syntheticpeptide amide: amorphous powder, 183 mg in yield. HPLC analysis:t_(R)=17.12 min, purity 98.9%, gradient 2% B to 22% B over 20 min; MS(MH⁺): expected mass 678.4, observed 678.5.

Example 58 Synthesis of compound (58):D-Phe-D-Phe-D-Leu-(δ-iPr)D-Orn-[ω(4-aminopiperidine-4-carboxylicacid)]—OH

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, Fmoc-D-Orn(Aloc)-OH, andN-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylic acid. The fully protectedresin bound peptide was synthesized manually starting from2-Chlorotrityl chloride resin (0.8 mmol). The attachment ofN-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylic acid to resin andsubsequent couplings were carried out according to the proceduredescribed in the synthesis of compound (38). The assembled peptideresin, Boc-D-Phe-D-Phe-D-Leu-D-Orn-ω(4-Aminopiperidine-4-carboxylicacid)-[2-Cl-Trt resin], was treated with Pd(PPh₃)₄ (4.5 mmol; Aldrich)in a mixture of CHCl₃/AcOH/NMM (80 ml, v/v/v=37:2:1) under Argonatmosphere at room temperature for 3 h for Aloc removal. SubsequentN-isopropylation was carried out according the procedure described inthe synthesis of compound (59). Final cleavage and preparative HPLCpurification were accomplished according to the procedure described inthe synthesis of compound (38). Final purified synthetic peptide amide:amorphous powder, 336 mg in yield. HPLC analysis: t_(R)=18.88 min,purity 98.9%, gradient 5% B to 25% B over 20 min; MS (MH⁺): expectedmolecular ion mass 708.4, observed 708.4.

Example 59 Synthesis of compound (59):D-Phe-D-Phe-D-Leu-(δ-iPr)D-Orn-[4-amidinohomopiperazine amide]

Synthesis was initiated from 0.9 g (0.5 mmol) of the peptide-resin:Z-D-Phe-D-Phe-D-Leu-D-Orn-homopiperazine-[carbamate Wang resin], whichwas prepared during the synthesis of compound (57) described above.

For isopropylation of the w-amino function of D-Orn at Xaa₄, the peptideresin was treated with a mixture of sodium triacetoxyborohydride (3mmol) and acetone (6 mmol) in TMOF (10 mL) over night at roomtemperature. The peptide resin was then treated with a solution ofCbz-OSu (6 mmol) in DMF (7 ml) for Cbz protection. The resin was washedwith DMF and DCM and dried in vacuo. The peptide was then cleaved fromthe resin by treatment with a solution of TFA/DCM (15 ml, v/v=1:1) atroom temperature for 2 hours. The resin was filtered and washed withTFA. The filtrate was evaporated in vacuo and the crude peptide (0.5mmol; Cbz-D-Phe-D-Phe-D-Leu-D-Orn(iPr,Cbz)-[homopiperazine amide]) wasobtained as an oil.

A portion of the above peptide (0.3 mmol) was continued for subsequentguanylation, cleavage and purification steps, which were carried outaccording to the procedure described in the synthesis of compound (57).Final purified synthetic peptide amide: amorphous powder, 166 mg inyield. HPLC analysis: t_(R)=18.71 min, purity 99.4%, gradient 2% B to22% B over 20 min; MS (MH⁺): expected mass 706.5, observed 706.5.

Example 60 Synthesis of compound (60):D-Phe-D-Phe-D-Leu-(δ-Me)D-Orn-[homopiperazine amide]

Synthesis was initiated from 0.2 mmol of the peptide intermediate,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(Me,Cbz)-[homopiperazine amide], which wasprepared during the synthesis of compound (57). The peptide washydrolyzed with a mixture of TMSOTf/TFA/m-cresol (10 ml, v/v/v=2:7:1)and the crude product was purified by preparative HPLC according to theprocedure described in the synthesis of compound (52). Final purifiedsynthetic peptide amide: amorphous powder, 98 mg in yield. HPLCanalysis: t_(R)=16.38 min, purity 99.6%, gradient 2% B to 22% B over 20min; MS (MH⁺): expected molecular ion mass 636.4, observed 636.5.

Example 61 Synthesis of compound (61):D-Phe-D-Phe-D-Leu-(δ-iPr)D-Orn-[homopiperazine amide]

Synthesis was initiated from 0.2 mmol of the peptide intermediate,Cbz-D-Phe-D-Phe-D-Leu-D-Orn(iPr,Cbz)-[homopiperazine amide], which wasprepared during the synthesis of compound (59). The peptide washydrolyzed with a mixture of TMSOTf/TFA/m-cresol (10 ml, v/v/v=2:7:1)and the crude product was purified by preparative HPLC according to theprocedure described in the synthesis of compound (52). Final purifiedsynthetic peptide amide: amorphous powder, 87 mg in yield. HPLCanalysis: t_(R)=18.41 min, purity 100%, gradient 2% B to 22% B over 20min; MS (MH⁺): expected molecular ion mass 664.5, observed 664.5.

Example 62 Synthesis of compound (62):D-Phe-D-Phe-D-Leu-D-Lys-[1,3-dioxolan-2-yl)methanamine amide]

Intermediate I-8 was prepared as described in Example 25. To a solutionof the acid, intermediate I-8 (150 mg, 0.20 mmol), the amineHNR_(a)R_(b), (1,3-dioxolan-2yl)methanamine (31 mg, 0.30 mmol) and DIEA(175 ul, 1.0 mmol) in DMF (5 mL) was added HBTU (113 mg, 0.3 mmol) at 0°C. After stiffing overnight from a starting temperature of 0° C. to roomtemperature, DMF was evaporated under reduced pressure. The residue wasstirred with 4N HCl in 1,4-dioxane (2.0 mL) at room temperature for 1hour. After removal of dioxane, the residue was dissolved in water andpurified by RP HPLC with a gradient of 10% MeCN/water to 60% MeCN/waterin 30 minutes to give pure synthetic peptide amide, compound (62) (44mg, 44% yield for the two steps) after evaporation of solvent. LC-MS:m/z=639 (MH⁺).

Example 63-75 Synthesis of Compounds (63)-(75)

Compounds (63)-(75) were prepared essentially as described in Example62, by substituting the appropriate amide in the coupling step asdetailed in U.S. Pat. No. 7,713,937. For example, for compound (63)2-(piperazin-1-yl)pyrimidine was used in the amide coupling step.

Example 76 Synthesis of Compound (76)

The amino acid derivatives, Boc-D-Phe-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH,Boc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, and Fmoc-Lys(Dde)-OH were used in thissynthesis. The fully protected resin bound peptide was synthesizedmanually starting from 2-chlorotrityl chloride resin (0.3 mmol; PeptideInternational). The attachment of the first amino acid to the resin wasachieved by treatment with a mixture of Fmoc-Lys(Dde)-OH (0.29 g, 0.5mmol; Novabiochem) and DIEA (0.35 mL, 2 mmol) in DCM (7 ml) at roomtemperature for 4 hours. The resin was washed with 3× DCM/MeOH/DIEA(v/v/v=17:2:1) and then treated with 25% piperidine in DMF for Fmocremoval. The subsequent peptide chain elongation was achieved byPyBOP/DIEA mediated single couplings with a 3-fold excess of amino acidderivatives, Fmoc-D-Orn(Boc)-OH and Boc-D-Leu-OH. The resulting peptideresin, Boc-D-Leu-D-Orn(Boc)-Lys(Dde)-[2-Cl-Trt resin], was treated with4% hydrazine in DMF three times for 3 min each to remove Dde. Thesubsequent peptide chain elongation was achieved by PyBOP/DIEA mediatedsingle couplings with a 3-fold excess of amino acid derivatives,Fmoc-D-Orn(Boc)-OH, Fmoc-D-Leu-OH, Fmoc-D-Phe-OH, and Boc-D-Phe-OH. TheFmoc group was removed with 25% piperidine in DMF. The fully assembledpeptide was cleaved from the resin by treatment with a mixture ofTFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperature for 90minutes. The resin was filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (0.3 mmol;D-Phe-D-Phe-D-Leu-D-Orn-[eLys(D-Orn-D-Leu-H)]—OH) was precipitated fromdiethyl ether.

For purification, the crude peptide (0.3 mmol) was dissolved in 2%acetic acid in H₂O (50 ml) and the solution was loaded onto an HPLCcolumn and purified using TEAP buffer system with a pH 5.2 (buffersA=TEAP 5.2 and B=20% TEAP 5.2 in 80% ACN). The compound was eluted witha linear gradient of buffer B, 10% B to 40% B over 60 minutes. Fractionswith purity exceeding 95% were pooled and the resulting solution wasdiluted with two volumes of water. The diluted solution was then loadedonto an HPLC column for salt exchange and further purification with aTFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1% TFA in 80%ACN/20% H₂O) and a linear gradient of buffer B, 2% B to 75% B over 25minutes. Fractions with purity exceeding 97% were pooled, frozen, anddried on a lyophilizer to yield the purified peptide as white amorphouspowder (396 mg). HPLC analysis: t_(R)=13.63 min, purity 99.7%, gradient10% B to 30% B over 20 min; MS (MH⁺): expected mass 895.5, observed895.6.

Example 77 Synthesis of Compound (77)

For the synthesis of compound (77) an additional amino acid residueD-Phe in the peptide resin intermediate,Boc-D-Phe-D-Leu-D-Orn(Boc)-Lys(Dde)-[2-Cl-Trt resin]. The resinintermediate was prepared by attachment of Fmoc-Lys(Dde)-OH to2-Chlorotrityl chloride resin followed by Fmoc removal and couplings ofamino acid derivatives Fmoc-D-Orn(Boc)-OH, Fmoc-D-Leu-OH, andBoc-D-Phe-OH. Final purified peptide: amorphous powder, 508 mg in yieldin a synthesis scale of 0.3 mmol HPLC analysis: t_(R)=18.90 min, purity100%, gradient 10% B to 30% B over 20 min; MS (MH⁺): expected molecularion mass 1042.4, observed 1042.7.

Examples 78-89 Synthesis of Compounds (78)-(89)

These compounds, (78) - (89), listed above, can be synthesized bymethods analogous to those used in the synthesis of compounds (76) and(77) described above.

Example 90 Synthesis of compound (90)D-Phe-D-Phe-D-Leu-D-Orn-[R/S-2-carboxymorpholine]—OH

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, Fmoc-D-Orn(Boc)-OH, and (R,S)-Fmoc-2-carboxymorpholine.The fully protected resin bound peptide was synthesized on a SYMPHONYMultiple Synthesizer (Protein Technology Inc.) starting from2-Chlorotrityl chloride resin (0.4 mmol; Novabiochem). The attachment ofthe first amino acid to the resin was achieved by treatment with amixture of (R,S)-Fmoc-2-carboxymorpholine (0.18 g, 0.5 mmol; NeoMPS) andDIEA (0.35 mL, 2 mmol) in DCM (7 ml) at room temperature for 4 hours.The resin was washed with 3× DCM/MeOH/DIEA (v/v/v=17:2:1) and 3× DCM.The subsequent peptide chain elongation was achieved by HBTU/DIEAmediated single couplings with a 3-fold excess of amino acidderivatives. The Fmoc group was removed by 25% piperidine in DMF. Forcleavage, the final peptide resin was treated with a mixture ofTFA/TIS/H2O (15 ml, v/v/v=95:2.5:2.5) at room temperature for 90minutes. The resin was filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (0.15 g,DPhe-DPhe-DLeu-DOrn-[R/S-2-carboxymorpholine]—OH) was precipitated fromdiethyl ether.

For purification, the above crude peptide (0.15 g) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 30 min, t_(R)=45% B. The fractions withpurity exceeding 97% were pooled, frozen, and dried on a lyophilizer togive the purified peptide as white amorphous powder (84 mg). Thecompound was a mixture of diastereoisomers as no attempt was made toseparate the two isomers, DPhe-DPhe-DLeu-DOrn-[R-2-carboxymorpholine]—OHand DPhe-DPhe-DLeu-DOrn-[S-2-carboxymorpholine]—OH. HPLC analysis:t_(R)=16.93 min (49.6%) and 17.34 min (50.4%), combined purity 100%,gradient 10% B to 30% B over 20 minutes.

Synthesis of Compounds (91)-(103)

Compounds (91)-(103) can be prepared by the above-described syntheticmethods and by other synthetic methods well known in the art.

Example 104 Synthesis of Compound (104)

Step 1: To a solution of H-D-Phe-D-Leu-OBn, I-4 (2.0 g, 4.27mmol),Boc-D-tert-Leu-OH (924 mg, 4.0 mmol) or Boc-D-tert-Butyl-D-Ala-OH (980mg, 4.0 mmol) and DIEA (1.0 mL, 6.0 mmol) in DMF was added HBTU (1.52 g)in three portions at 0° C. over a 10 minute period. The mixture wasstirred from 0° C. to room temperature overnight. After evaporation ofDMF, the residue was extracted between ethyl acetate and saturatedsodium bicarbonate. The organic layer was dried over sodium sulfate andevaporated to dryness. The residue was purified by column chromatographywith 10% to 60% ethyl acetate/hexanes to give pure product. (96% yieldfor R=tert-butyl and 90% yield for R=neopentyl). The dipeptide benzylester was hydrogenated with palladium on carbon (10%, 300 mg) with ahydrogen balloon in MeOH for 3 hours. The mixture was filtered throughcelite and evaporated to give the tripeptide product (91% yield forR=tert-butyl and 100% yield for R=neopentyl).

Step 2: To a solution of Fmoc-D-Lys(Boc)-OH (468 mg, 1.0 mmol), benzyl4-(benzyloxycarbonylamino)piperidine-4-carboxylate (368 mg, 1.0 mmol)and DIEA (520 uL, 3.0 mmol) in DMF was added HBTU (474 mg, 1.25 mmol) inthree portions at 0° C. over 10 minutes. The mixture was stirred from 0°C. to room temperature overnight. After evaporation of DMF, the residuewas extracted between ethyl acetate and saturated sodium bicarbonate.The organic layer was dried over sodium sulfate and evaporated todryness. The residue was purified by column chromatography with 30% to60% ethyl acetate/hexanes to give pure product (760 mg, 93%, m/e=819(MH)⁺). The intermediate obtained (760 mg) was dissolved in DCM and thenpyrrolidine (4 mL) was added. After stiffing at room temperature for 1hour, the DCM was evaporated from the mixture. The residue was dissolvedin MeOH and purified by a C18 column with 5% to 60% MeCN/water in 30mins Pure fractions were combined and lyophilized overnight to give thedesired product (580 mg).

Step 3: To a solution of the tripeptide obtained in step 1 (R=neopentyl,152 mg, 0.30 mmol), the product obtained in step 2 (179 mg, 0.30 mmol)and DIEA (170 uL, 1.0 mmol) in DMF was added HBTU (152 mg, 0.40 mmol) at0° C. The mixture was stirred from 0° C. to room temperature for 2hours. After evaporation of DMF, the residue was dissolved in MeOH andpurified by a C18 column chromatography with a gradient of 30% to 100%MeCN/water in 40 minutes. Pure fractions were combined and lyophilizedovernight to give the desired product (180 mg, 55%, m/e=1084 (M+H)).

Step 4: A solution of the product obtained in step 3 (82 mg) in MeOH washydrogenated with palladium on carbon (10%, 20 mg) under a hydrogenballoon for 1 hour. The mixture was filtered through celite andevaporated to dryness. The residue obtained was stirred with TFA/DCM (4mL, 1:1 v/v) at room temperature for 1 hour. After evaporation ofsolvent, the residue was purified by C18 column chromatography byelution with a gradient of 0% to 100% MeCN/water in 40 minutes. Purefractions were combined and lyophilized overnight to give compound (104)(39 mg, 78% yield, LC-MS: m/z=661 (MH)⁺).

Example 105 Synthesis of Compound (105)

Step 1: To a solution of H-D-tert-Leu-D-Phe-D-Leu-OH synthesizedaccording to step 1 in the synthesis of compound (104) (R=tert-butyl,491 mg, 1.0 mmol), D-Lys(Boc)-OAll (323 mg, 1.0 mmol) and DIEA (440 uL,2.5 mmol) in DMF was added HBTU (400 mg, 0.40 mmol) at 0° C. The mixturewas stirred from 0° C. to room temperature for 3 hours. Afterevaporation of DMF, the residue was precipitate in water (200 mL) andfiltered to give the tetrapeptide intermediate (0.79 g, 99% yield,m/z=761, (MH)⁺). To a solution of the tetrapeptide allyl ester (0.79 g,1.04 mmol) in MeCN was added palladium tetrakis-(triphenylphosphine)(120 mg) and pyrrolidine (345 uL) at 0° C. After stirring at roomtemperature overnight, the acetonitrile was evaporated from the mixture.The residue was purified by C18 column chromatography with a gradient of30% to 90% MeCN/water. Pure fractions were combined and lyophilized togive the desired product (0.41 g, 57%), m/z=720 (MH)⁺.

Step 2: To a solution of the tetrapeptide obtained in Step 1 (180 mg,0.25 mmol), benzyl 4-(benzyloxycarbonyl-amino)piperidine-4-carboxylate(101 mg, 0.25 mmol) and DIEA (250 uL, 1.4 mmol) in DMF was added HBTU(140 mg, 0.37 mmol) at 0° C. The mixture was stirred from 0° C. to roomtemperature overnight. After evaporation of DMF, the residue waspurified purified by C18 column chromatography with a gradient of 25% to80% MeCN/water. Pure fractions were combined and lyophilized to give thedesired product (0.37 g, 100%), m/z=1071 (MH)⁺.

Step 3: A solution of the product obtained in Step 2 (0.37 g) in TFA/DCMwas stirred at room temperature for 1 hour. After evaporation ofsolvent, the residue was extracted with ethyl acetate and saturatedsodium bicarbonate. The organic layer was dried over sodium sulfate andevaporated to dryness. The residue (0.20 g) was hydrogenated withpalladium on carbon (10%, 50 mg) in MeOH under a hydrogen balloon for 1hour. The mixture was filtered through celite and evaporated to dryness.The residue was dissolved in MeOH and purified by C18 columnchromatography with a gradient of 5% to 60% MeCN/water. Pure fractionswere combined and lyophilized to give compound (105) (50 mg, 27%),m/z=647 (MH)⁺.

Example 106 Inhibition of cAMP Production by Stimulation of EndogenousMouse Kappa-Opioid Receptor in R1.G1 Cells

Potency of the synthetic peptide amides as kappa-opioid receptoragonists was determined by measuring the inhibition offorskolin-stimulated adenylate cyclase activity. R1.G1 cells (a mousethymoma cell line that expresses only the kappa-opioid receptor and noother opioid receptor subtype) were first exposed to forskolin (toinduce cAMP) plus the synthetic peptide amide at the test concentration.After incubation, the cAMP level in the challenged R1.G1 cells wasdetermined using a time resolved fluorescence resonance energy transfer(TR-FRET)-based cAMP immunoassay (LANCE™, Perkin Elmer). The detailedmethod is described below:

Mouse R1.G1 cells (ATCC, Manassas, Va.) were grown in suspension in highglucose-DMEM (Dulbecco's Modified Eagle's Medium, Cellgro, Herndon, Va.)containing 10% horse serum and 2% glutaMax (Invitrogen, Carlsbad.Calif.) without added antibiotics. On the day of the experiment, cellswere spun at 1,000 rpm for 5 minutes at room temperature and then washedonce with HBSS (Hanks' Balanced Saline Solution, Invitrogen, Carlsbad,Calif.). Cells were then spun again and resuspended in stimulationbuffer (HBSS with 0.05% FAF-BSA (Fatty acid-free bovine serum albumin,Roche Applied Science, Indianapolis, Ind.), 5 mM HEPES) to 2 millioncells per ml. Antibody supplied with the LANCE™ cAMP immunoassay kit wasthen added to the cells according to the manufacturer's instructions,and 12,000 cells per well were then added to the wells containingforskolin to a predetermined fixed final concentration (typically about2.5 uM) and the previously determined amount of the synthetic peptideamide to be tested.

The synthetic peptide amides were tested in a range of concentrations todetermine potency. Cells were incubated with the synthetic peptide amideplus forskolin for about 20 minutes at room temperature. Afterincubation, cells are lysed by adding 12 ul of detection mix as suppliedwith the LANCE™ kit, followed by incubation for one hour at roomtemperature. Time resolved fluorescence was read using a 330-380 nmexcitation filter, a 665 nm emission filter, dichroic mirror 380, andZ=1 mm A standard curve for cAMP concentration in this assay permitteddetermination of the amount of cAMP present in each well. A curve wasproduced by plotting synthetic peptide amide concentration against cAMPlevels in the test cells, and subjected to non-linear regression using afour-parameter curve fitting algorithm to calculate the EC₅₀, theconcentration of the synthetic peptide amide required to produce 50% ofthe maximal suppression of cAMP production by the synthetic peptideamide.

Example 107 Potency of Synthetic Peptide Amides on the Human KappaOpioid Receptor

Human Embryonic Kidney cells (HEK-293 cells, ATCC, Manassas, Va.) in 100mm dishes were transfected with transfection reagent, Fugene6 (RocheMolecular Biochemicals) and DNA constructs in a 3.3 to 1 ratio. The DNAconstructs used in the transfection were as follows: (i) an expressionvector for the human kappa opioid receptor, (ii) an expression vectorfor a human chimeric G-protein, and (iii) a luciferase reporterconstruct in which luciferase expression is induced by the calciumsensitive transcription factor NFAT.

The expression vector containing the human kappa opioid receptor wasconstructed as follows: The human OPRK1 gene was cloned from humandorsal root ganglion total RNA by PCR and the gene inserted intoexpression vector pcDNA3 (Invitrogen, Carlsbad, Calif.) to constructhuman OPRK1 mammalian expression vector pcDNA3-hOPRK1.

To construct the human chimeric G-protein expression vector, thechimeric G-protein Gαqi5 was first constructed by replacing the last 5amino acids of human Gαq with the sequence of the last 5 amino acids ofGai by PCR. A second mutation was introduced to this human Gαqi5 gene atamino acid position 66 to substitute a glycine (G) with an aspartic acid(D) by site-directed mutagenesis. This gene was then subcloned into amammalian expression vector pcDNA5/FRT (Invitrogen) to yield the humanchimeric G-protein expression vector, pcDNA5/FRT-hGNAq-G66D-i5.

To prepare the luciferase reporter gene construct, synthetic responseelements including 3 copies of TRE(12-O-tetradecanoylphorbol-13-acetate-responsive elements) and 3 copiesof NFAT (nuclear factor of activated T-cells) were incorporated upstreamof a c-fos minimal promoter. This response element and promoter cassettewas then inserted into a luciferase reporter gene vector pGL3-basic(Promega) to construct the luciferase reporter gene plasmid constructpGL3b-3TRE-3NFAT-cfos-Luc.

The transfection mixture for each plate of cells included 6 microgramspcDNA3-hOPRK1, 6 micrograms of pcDNA5/FRT-hGNAq-G66D-i5, and 0.6micrograms of pGL3b-3TRE-3NFAT-cfos-Luc. Cells were incubated for oneday at 37° C. in a humidified atmosphere containing 5% CO₂ followingtransfection, and plated in opaque 96-well plates at 45,000 cells perwell in 100 microliters of medium. The next day, test and referencecompounds were added to the cells in individual wells. A range ofconcentrations of test compounds was added to one set of wells and asimilar range of concentrations of reference compounds was added to aset of control wells. The cells were then incubated for 5 hours at 37°C. At the end of the incubation, cells were lysed by adding 100microliters of detection mix containing luciferase substrate (AMP (22ug/ml), ATP (1.1 mg/ml), dithiothreitol (3.85 mg/ml), HEPES (50 mM finalconcentration), EDTA (0.2 mg/ml), Triton N-101 (4 ul/ml), phenylaceticacid (45 ug/ml), oxalic acid (8.5 ug/ml), luciferin (28 ug/ml), pH 7.8).Plates were sealed and luminescence read within 30 minutes. Theconcentration of each of the compounds was plotted against luminescencecounts per second (cps) and the resulting response curves subjected tonon-linear regression using a four-parameter curve-fitting algorithm tocalculate the EC₅₀ (the concentration of compound required to produce50% of the maximal increase in luciferase activity) and the efficacy(the percent maximal activation compared to full induction by any of thewell-known kappa opioid receptor agonists, such as asimadoline(EMD-61753: See Joshi et al., 2000, J. Neurosci. 20(15):5874-9), orU-69593: See Heidbreder et al., 1999, Brain Res. 616(1-2):335-8).

The EC₅₀ values were obtained from the cAMP inhibition assay withsynthetic peptide amides synthesized according to the present inventionand tested on mouse kappa opioid receptor (mKOR EC₅₀ values from0.005-100 nM) and on the human kappa opioid receptor (hKOR EC₅₀ valuesfrom 0.001-1 nM) as described above.

Synthetic peptide amides of the invention were tested in a similar assayfor potency on the human mu opioid receptor. Each compound tested had anEC₅₀ for the human mu opioid receptor greater than or equal to 1 μM. Thesynthetic peptide amide (53) was tested in a similar assay for potencyon the human mu opioid receptor. The compound had an EC₅₀ for the humanmu opioid receptor greater than or equal to 1 μM.

Example 108 Membrane Permeability of the Synthetic Peptide Amides

The Caco-2 cell line is a human colon adenocarcinoma cell line thatdifferentiates in culture and is used to model the epithelial lining ofthe human small intestine. Compounds of the present invention can betested in a membrane permeability assay using the TC7 subclone of Caco-2in a standard assay (Cerep, Seattle, Wash.). Briefly, the apparentpermeability coefficient (P_(app)) can be determined in theapical-to-basolateral (A-B) direction across cell monolayers cultured on96-well polycarbonate membrane filters.

Compounds were tested at a concentration of 10 μM at pH 6.5 in 1% DMSO,with the recipient side maintained at pH 7.4. The assay plate wasincubated for 60 minutes at 37° C. with gentle shaking. Samples weretaken at time zero from the donor side and at the end of the incubationperiod from both the donor and recipient sides. Samples were analyzed byHPLC-MS/MS. The P_(app-)value (expressed as 10⁻⁶ cm/sec) was thencalculated based on the appearance rate of compound in the recipientside. The P_(app) was calculated with the equation:

$P_{app} = {\frac{1}{S \cdot C_{0}}\left( \frac{Q}{T} \right)}$

where P_(app) is the apparent permeability; S is the membrane surfacearea, C₀ is the donor concentration at time 0, and dQ/dt is the amountof drug transported per time. Four reference compounds (labetalol,propranolol, ranitidine, and vinblastine) were concurrently tested toensure the validity of the assay, as well as asimadoline, which ispurported to be a peripherally acting kappa opioid. Results are shown inTable I (below).

Compounds that exhibit low permeability in this type of assay arebelieved to have reduced potential for crossing the blood-brain barrierin vivo, since high passive permeability appears to be a key feature ofCNS-acting drugs (Mahar Doan et al. Passive permeability andP-glycoprotein-mediated efflux differentiate central nervous system(CNS) and non-CNS marketed drugs. J Pharmacol Exp Ther.2002;303:1029-37).

TABLE I Membrane permeability Compound Mean Permeability (cm⁻⁶/sec) (1)<0.10 (3) <0.02 (6) <0.02 Asimadoline 37.5 Labetalol 9.9 Propranolol53.8 Ranitidine 0.5 Vinblastine <0.2

Example 109 Inhibition of Cytochrome P₄₅₀ Oxidases

Inhibition of cytochrome P₄₅₀ oxidase isozymes CYP1A, CYP2C9, CYP2C19,CYP2D6 and CYP3A4 by synthetic peptide amide compounds of the inventionwas determined according to the following methods performed by Cerep(Seattle, Wash.):

In the cytochrome P₄₅₀ CYP1A assay, human liver microsomes (0.2 mg/mlprotein) were incubated for 15 minutes at 37° C. with 10 μM testcompound, 1 μM ethoxyresorufin, 1.3 mM NADP, 3.3 mM glucose-6-phosphateand 0.4 U/ml glucose-6-phosphate dehydrogenase. In the absence of testcompound, the ethoxyresorufin added as substrate is oxidized toresorufin, and in the presence of an inhibitor of the CYP isozyme, theamount of resorufin produced is reduced. Furafylline was used as areference inhibitor.

The cytochrome P₄₅₀ CYP2C9 assay reaction mixture containing human livermicrosomes (0.2 mg/ml protein) was incubated for 15 minutes at 37° C.with 10 μM test compound, 10 μM tolbutamide, 1.3 mM NADP, 3.3 mMglucose-6-phosphate and 0.4 U/ml glucose-6-phosphate dehydrogenase. Inthe absence of test compound, the tolbutamide is oxidized to4-hydroxytolbutamide, and in the presence of an inhibitor of the CYPisozyme, the amount of 4-hydroxytolbutamide produced is reduced.Sulfaphenazole (IC₅₀: 0.35 μM) was the reference inhibitor.

For the cytochrome P₄₅₀ CYP2C19 assay, human liver microsomes (0.2 mg/mlprotein) were incubated for 15 minutes at 37° C. with 10 μM testcompound, 10 μM omeprazole, 1.3 mM NADP, 3.3 mM glucose-6-phosphate and0.4 U/ml glucose-6-phosphate dehydrogenase. In the absence of testcompound, the omeprazole is oxidized to 5-hydroxy-omeprazole, and in thepresence of an inhibitor of the CYP isozyme, the amount of5-hydroxy-omeprazole produced is reduced. Oxybutinin (IC₅₀: 7.1 μM) wasthe reference inhibitor.

The cytochrome P₄₅₀ CYP2D6 assay reaction containing human livermicrosomes (0.2 mg/ml protein) was incubated for 15 minutes at 37° C.with 10 μM test compound, 5 μM dextromethorphan, 1.3 mM NADP, 3.3 mMglucose-6-phosphate and 0.4 U/ml glucose-6-phosphate dehydrogenase. Inthe absence of test compound, the dextromethorphan is oxidized, and inthe presence of an inhibitor of the CYP isozyme, the amount of oxidationproduct is reduced. Quinidine (IC₅₀: 0.093 μM) was the referenceinhibitor.

For the cytochrome P₄₅₀ CYP2C19 assay, human liver microsomes (0.2 mg/mlprotein) were incubated for 20 minutes at 37° C. with 10 μM testcompound, 5 μM midazolam, 1.3 mM NADP, 3.3 mM glucose-6-phosphate and0.4 U/ml glucose-6-phosphate dehydrogenase. In the absence of testcompound, the midazolam is oxidized, and in the presence of an inhibitorof the recombinant isozyme, the amount of oxidation product is reduced.The oxidation product is determined from the area under the curve afterHPLC-MS/MS separation. Ketoconazole (IC₅₀: 0.55 μM) was the referenceinhibitor.

In each assay, the percent inhibition of the cytochrome P₄₅₀ CYP P₄₅₀isozyme was determined as one hundred times the ratio of (1−minus theamount of product in the sample in the presence of the test compound)divided by the amount of product in the sample containing untreatedisozyme. The results of duplicate assays (expressed as percent remainingCYP activity) are shown in Table II.

TABLE II Percent Activity of Cytochrome P₄₅₀ CYP Isozymes Compound (54)(54) P₄₅₀ isozyme (1) (3) (6) Expt 1 Expt 2 CYP1A 89.8 93.1 89.5 97.195.8 CYP2C9 93.2 97.4 92.1 99.2 98.2 CYP2C19 98.5 103.2 97.2 94.1 97.8CYP2D6 96.0 99.5 93.9 98.4 98.2 CYP3A4 92.5 94.3 93.6 94.7 95.9

Example 110 Pharmacokinetics of Compound (2) in Rat

To determine brain to plasma concentration ratios of compound (2), agroup of 6 conscious jugular vein catheterized rats were administered 3mg/kg of peptide over a 5 minute infusion period into the jugular veincatheter. Thirty, 60 and 180 minutes following the start of infusion,blood samples were collected from two animals at each time point byterminal cardiac puncture and whole brains were rapidly removed. Plasmawas isolated by centrifugation. Tandem liquid chromatography massspectrometry (LC-MS/MS) was used to quantify the concentration of drugin rat plasma and brain. Results are shown in FIG. 1.

Example 111 Pharmacokinetics of Compound (6) in Mice

A single bolus of the synthetic peptide amide compound was administeredby subcutaneous injection to ICR mice (n=6, males, body wt 23-37 g,Charles River, Wilmington, Mass.) and plasma samples taken at 5, 10, 15,20, 30 60, 90, 120, and 180 minutes post-injection. FIG. 2 shows theresults obtained after subcutaneous injection of a 1 mg/kg dose ofcompound (6) in ICR mice. The “half life” for this study was determinedas the time required for the plasma concentration to fall by 50% aftermaximum concentration in the plasma was achieved; the computedelimination half-life, based on the elimination rate constant of theslowest elimination phase, is expected to be longer. See Table IIIbelow.

Example 112 Pharmacokinetics of Synthetic Peptide Amide Compounds (52)and (6) in Cynomolgus Monkeys

A single bolus of the synthetic peptide amide compound was administeredby intravenous injection to cynomolgus monkeys (n=4 males, body wt 3-5kg, SNBL USA, Ltd., Everett, Wash.), and plasma samples taken at 5, 10,15, 20, 30 60, 90 120, and 180 minutes post-injection. The “half life”was determined as the time required for the plasma concentration to fallby 50% after maximum concentration in the plasma was achieved.Cynomolgus monkeys were injected with a single intravenous dose ofsynthetic peptide amide compound (52) according to the protocol detailedin Example 52, and the half life of plasma concentration determined.

Results are shown in Table III.

TABLE III In vivo half life of synthetic peptide amide compounds (6) and(52) Cynomolgus Monkeys (6) (52) Administration Route intravenousintravenous Half Life (min) 58.6 69

Persistence of compound (3) in the plasma of cynomolgus monkeys afterintravenous administration of a bolus of 0.56 mg/kg is shown in FIG. 3.

Example 113 Acetic Acid-Induced Writhing Assay in Mice

This test identifies compounds which exhibit analgesic activity againstvisceral pain or pain associated with activation of low pH-sensitivenociceptors [see Barber and Gottschlich (1986) Med. Res. Rev. 12:525-562; Ramabadran and Bansinath (1986) Pharm. Res. 3: 263-270].Intraperitoneal administration of dilute acetic acid solution causes awrithing behaviour in mice. A writhe is defined as a contraction of theabdominal muscles accompanied by an extension of the forelimbs andelongation of the body. The number of writhes observed in the presenceand absence of test compounds is counted to determine the analgesicactivity of the compounds.

Each day a writhing assay was performed, a vehicle control group of mice(n=6−8) that were treated identically to the test group (except thattest compound was omitted from the injection dose) was always includedand the average total number of writhes in this group used as theabsolute reference point defining 0% decrease in pain perception for allother mice receiving a test compound on that day. Specifically, thetotal number of writhes of each mouse receiving the test compound wasconverted to % decrease in pain perception according to the followingequation:

${\% \mspace{14mu} {decrease}\mspace{14mu} {in}\mspace{14mu} {pain}\mspace{14mu} {perception}} = {\frac{\left( {W_{v} - W_{c}} \right)}{W_{v}} \times 100}$

Where W_(v) is the mean number of writhes in vehicle-treated group andW_(c) is the number of writhes in compound-treated mouse. The data wereanalyzed using the 2-parameter Hill's equation (a.k.a. Emax model),where Emax is assumed to be 100% antinociperception (i.e., no writhesover the 15 min post-acetic acid administration).

Male ICR mice, 23-37 grams in weight, were weighed and placed inindividual observation chamber (usually a 4000 ml glass beaker) with afine layer of SANI-CHIPS rodent bedding at the bottom. To determine theactivity and potency of test compounds, different doses of the compoundsolution or vehicle were injected subcutaneously in the back of the neck15 or 180 minutes prior to administration of acetic acid solution. Afteradministration of the compound or vehicle control, mice were returned totheir individual observation chambers awaiting the intraperitonealadministration of acetic acid solution. Fifteen minutes or three hourslater, according to the interval time defined in each experiment betweencompound delivery and acetic acid injection, a dose corresponding to 10ml/kg of a 0.6% (v/v) acetic acid solution was then injectedintraperitoneally (i.p.) into the right lower quadrant of the abdomenImmediately after the injection, the mouse was returned to itsobservation chamber and the recording of the number of writhes begunimmediately. The number of writhes was counted over a 15-min periodstarting from the time of acetic acid injection, the data beingcollected over three separate 5 minute time periods (0-5 min, 5-10 min,and 10-15 min)

The data were reported as ED₅₀, and Hill coefficient. The ED₅₀ isexpressed either as mean±standard error of the mean (sem) (ED₅₀+/−sem)or as geometric mean with 95% confidence intervals (95% CI) usingt-scores. The Hill coefficient is expressed as the arithmetic mean±semcalculated from the values obtained from the animals. Results forcompound (2) are shown in FIG. 4 (solid circles).

For dose-response analysis, raw data were converted to % maximumpossible effect (% MPE) using the formula: % MPE=((testscore−vehicle-treated score)/(0−vehicle-treated score))×100. Raw datawere analyzed using a one-way ANOVA followed by Dunnett's post-tests.The dose which elicited 50% attenuation of hypersensitivity (ED₅₀) wasdetermined using linear regression analysis. Compounds were administeredby the intravenous route. Table IV summarizes the results of theseexperiments.

TABLE IV Effects of Compounds (2) and (5) on Acetic Acid-InducedWrithing in Mice. ED₅₀ % MPE % MPE % MPE Com- (mg/kg, iv, (180 min (240min (300 min pound 15 min post-dose) post-ED₉₀) post-ED₉₀) post-ED₉₀)(2) 0.07 (0.06-0.1) 77 ± 5%  81 ± 4% 84 ± 4% (5) 0.01 (0.01-0.02) 54 ±10% NT NT NT = not tested

A dose response for compound (2) in the acetic acid-induced writhingmodel in mice was generated using 0.01, 0.03, 0.1 and 0.3 mg/kgadministered intravenously as described above. Using the above method alinear dose response relationship was determined for compound (2) fordoses ranging from 0.01 mg/kg to 0.3 mg/kg, as shown in FIG. 5.

Example 114 Inhibition of Locomotion in Mice to Measure Sedation byCompounds After Subcutaneous Injection (Locomotion Reduction Assay)

Compounds which exhibit sedative activity inhibit the spontaneouslocomotion of mice in a test chamber. To determine the potentialsedative effect of test compounds, the extent of locomotion after theadministration of the test compound or vehicle control can be determinedand compared with a specialized apparatus designed for this purpose(Opto-Varimex Activity Meter). At the start of each experiment, eachmouse was weighed and examined to determine good health. To determinethe activity and potency of compounds, different doses of the compoundsolution or vehicle were injected subcutaneously 15 or 180 minutes priorto initiation of data collection. The subcutaneous injection wasperformed in the back of the neck of the mouse, pinched in a “tent” toallow proper access for the syringe needle. After injection, each animalwas placed individually in Plexiglas boxes (43 cm×43 cm) inside theOpto-Varimex Activity Meter apparatus. Before the animal was placed inthe apparatus, a thin layer of SANI-CHIPS rodent bedding was placed onthe bottom of the Plexiglas box to provide a comfortable environment.Each Opto-Varimex Activity Meter apparatus was then turned on and dataacquisition begun by the ATM3 Auto-Track System. The data were processedand results expressed in the same way as described for the writhingassay data above.

Example 115 Analgesic vs. Sedative Effects of Synthetic Peptide Amide(54):D-Phe-D-Phe-D-Leu-D-Orn-[4-(4,5-dihydro-1H-imidazol-2-yl)homopiperazineamide]

Inhibition of acetic acid-induced writhing is an indication of ananalgesic effect (also called an antinociceptive effect). A reduction inlocomotion can be used as a measure of a general sedative effect. TheED₅₀ determined in the acetic acid-induced writhing assay in IRC micewas 74 μg/kg [with a 95% confidence interval of 49-99 μg/kg] whensynthetic peptide amide (54) was delivered subcutaneously. The ED₅₀value determined in the inhibition of locomotion assay was 3172 μg/kg[with a 95% confidence interval of 1810-4534 μg/kg] for the samesynthetic peptide amide delivered subcutaneously. See FIG. 6. Thetherapeutic ratio of the analgesic effect over the sedative effect isthe fold higher ED₅₀ required to achieve a sedative effect as comparedto the ED₅₀ required to achieve an analgesic effect. Thus, compound (54)exhibits a (3172/74) fold ratio, i.e. 42.86 fold. Thus, the therapeuticratio for compound (54) is approximately 43 fold.

Example 116 Analgesic vs. Sedative Effects of Synthetic Peptide Amide(3)

Inhibition of acetic acid-induced writhing by a compound is anindication of an analgesic effect (also called an antinociceptiveeffect). Similarly, a reduction in locomotion caused by administrationof the compound can be used as a measure of its general sedative effect.

The ED₅₀ determined in the acetic acid-induced writhing assay in ICRmice was 52 μg/kg when synthetic peptide amide (3) was administeredsubcutaneously as described in Example 113 and shown in FIG. 4 (solidcircles). The ED₅₀ value determined in the inhibition of locomotionassay as described in Example 114 was 2685 μg/kg for the same syntheticpeptide amide administered subcutaneously. See FIG. 4 (solid squares).The therapeutic ratio of the analgesic effect over the sedative effectis the fold higher ED₅₀ required to achieve a sedative effect ascompared to the ED₅₀ required to achieve an analgesic effect. Thus,compound (3) exhibits a (2685/52) fold ratio, i.e. 51.6 fold. Thus, thetherapeutic ratio is approximately 52 fold for compound (3).

Example 117 Synthetic Peptide Amide Pharmacokinetcs in Monkeys

Samples were administered to male monkeys, Macaca fascicularis (SNBLUSA, Ltd., Everett, Wash., purpose-bred cynomolgus monkeys. Closelyrelated to humans, both phylogenetically and physiologically) aged 3-7years and weighing 3-5 kilograms. Samples were administered in asuperficial vein of the arm or leg (e.g. brachial, or saphenous) in 0.9%saline for injection, USP (Baxter Healthcare, Deerfield, Ill.) asfollows: A cassette sample containing 0.4 mg of compound (52) of thepresent invention and 0.4 mg of each of nine other compounds (for atotal dose of 4 mg) was prepared in 2 ml 0.9% saline for injection,providing a concentration of 0.2 mg/ml of each of the ten compounds.Exactly 2 ml was administered as an intravenous bolus to the testanimal, resulting in a total dose level of 0.8 to 1.3 mg/kg depending onthe body weight of the individual animal. The intravenous injection wasfollowed by a 1 ml flush with 0.9% saline for injection. Blood samplesof 0.6 ml were collected by venipuncture from a peripheral vein at 2, 5,10, 15 and 30 minutes post dose injection, and then at 1, 2 and 4 hours.Each sample was placed in a pre-chilled glass test tube containinglithium heparin and immediately chilled on ice. Plasma was collectedafter centrifugation at 2,000 g for fifteen minutes at 2-8° C. Theplasma layers of each sample were transferred to polypropylene tubes andstored frozen at −60° C. or lower until assayed. One hundred microliteraliquots of thawed plasma were spiked with 5 microliters of a 400 ng/mlsolution of an appropriate internal standard (in this case a knownsynthetic peptide amide compound) in 0.1% TFA, and the proteins wereprecipitated with 100 microliters of 0.1% TFA in acetonitrile. Thesamples were centrifuged at 1000×g for 5 minutes and the supernatantsanalyzed by LC-MS. LC-MS analysis was performed on a Finnigan LCQ Decamass spectrometer interfaced to a Surveyor HPLC system (Thermo ElectronCorporation, Waltham, Mass., USA). HPLC analysis was performed on2.1×150 mm C18 reversed phase columns with a gradient of 0.01% TFA inacetonitrile in 0.01% TFA in water. Mass detection was performed in theselected reaction monitoring mode (SRM). Quantitation was performedagainst a calibration curve of the analyte in blank cynomolgus monkeyplasma using the same internal standard. Data analysis and theextraction of pharmacokinetic parameters were performed with the programPK Solutions 2.0 (Summit Research Services, Ashland, Ohio, USA). Resultsfor compound (52):D-Phe-D-Phe-D-Leu-D-Orn-[4-(N-methyl)amidino-homopiperazine amide] areshown in FIG. 7.

Example 118 Spinal Nerve Ligation (SNL) Model

The SNL model (Kim and Chung 1992) was used to induce chronicneuropathic pain. The rats were anesthetized with isoflurane, the leftL5 transverse process was removed, and the L5 and L6 spinal nerves weretightly ligated with 6-0 silk suture. The wound was then closed withinternal sutures and external staples. Fourteen days following SNL,baseline, post-injury and post-treatment values for non-noxiousmechanical sensitivity were evaluated using 8 Semmes-Weinstein filaments(Stoelting, Wood Dale, Ill., USA) with varying stiffness (0.4, 0.7, 1.2,2.0, 3.6, 5.5, 8.5, and 15 g) according to the up-down method (Chaplanet al. 1994). Animals were placed on a perforated metallic platform andallowed to acclimate to their surroundings for a minimum of 30 minutesbefore testing. The mean and standard error of the mean (SEM) weredetermined for each paw in each treatment group. Since this stimulus isnormally not considered painful, significant injury-induced increases inresponsiveness in this test are interpreted as a measure of mechanicalallodynia. The dose which elicited 50% attenuation of mechanicalhypersensitivity (ED₅₀) was determined using linear regression analysis.Compound (2) was administered by the intravenous route. FIG. 8summarizes the results of these experiments. The calculated ED₅₀ forcompound (2) in this model was 0.38 mg/kg (0.31-0.45; 95% confidenceinterval).

Example 119 Ocular Analgesia Induced by Compounds (2), (3) and (4)

Ocular analgesia was evaluated by instilling five volumes of the testcompound, 50 microliters each in physiological saline, at theconcentration to be tested into the right eye of naive albino NewZealand strain rabbits within a period of twenty minutes. Fifteenminutes after the last instillation of the test compound, each animalwas administered a single instillation of 30 microliters of 10 mg/mlcapsaicin (33 mM) in the treated eye. Capsaicin is known to inducecorneal pain. Corneal pain was evaluated by measurement of the palpebralopening measured in millimeters using a transparent ruler over thetreated and untreated eyes. In this animal model, the reduction in sizeof the palpebral opening after instillation of capsaicin is anindication of the degree of ocular pain. Thus, any observed restoration(increase) in size of the palpebral opening after treatment with testcompound is taken as a measure of relief from capsaicin-induced ocularpain.

These evaluations were performed before treatment with the test compound(pre-test), immediately prior to the instillation of capsaicin, and then1, 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes following theinstillation of capsaicin. Table V shows the mean of palpebral openingmeasurements (relative to the untreated eye expressed as percent ofcontrol) averaged over the period from 10-30 minutes after capsaicininstillation in rabbits pre-instilled with a kappa opioid agonist of theinvention and after preinstillation with a standard concentration ofdiltiazem, a benzothiazepine calcium channel blocker with localanesthetic effects. See Gonzalez et al., (1993) Invest. Ophthalmol. Vis.Sci. 34: 3329-3335.

TABLE V Effect of compounds (2), (3) and (4) in reducing ocular painTime Mean SEM Compound (post capsaicin) (% Control) (% Control) None(Saline) 10-30 min. 61.2 6.5 Diltiazem at 10 mM 10-30 min. 74.6 5.5 (2)at 10 mg/ml 10-30 min. 82.7 5.3 (3) at 10 mg/ml 10-30 min. 76.0 5.2 (4)at 10 mg/ml 10-30 min. 56.7 8.4 Mean is of five animals; SEM: Standarderror of the mean

Example 120 Dose Response of Compound (2) in Capsaicin-Induced OcularPain

Ocular analgesia induced by Compound (2) at several concentrationsinstilled into the right eye of naive albino New Zealand strain rabbitswas evaluated as described above. Results were compared with analgesiainduced by 10 mg/ml morphine (a non-selective opioid agonist) as asystemic active control, and with 10 mM diltiazem as a topicalactive-control in the same experiment and under the same conditions.Table VI below shows the accumulated results.

TABLE VI Dose-Response of Compound (2) in Capsaicin-Induced Ocular PainTime Mean SEM Compound (post capsaicin) (% Control) (% Control) Morphineat 10 mg/ml 10-30 min. 74.8 11.1 Diltiazem at 10 mM 10-30 min. 77.6 7.4(2) at 1 mg/ml 10-30 min. 60.5 9.9 (2) at 10 mg/ml 10-30 min. 56.6 9.3(2) at 25 mg/ml 10-30 min. 75.5 7.1 (2) at 50 mg/ml 10-30 min. 87.5 4.8Mean is of ten animals; SEM: Standard error about the mean

Example 121 Effect of Compound (2) in a Rat Pancreatitis Model

Chronic pancreatic inflammation was induced in rats by intravenousadministration of dibutyltin dichloride (DBTC, Aldrich Milwaukee, Wis.)dissolved in 100% ethanol at a dose of 8mg/kg under isofluoraneanesthesia (2-3 liters/min, 4%/vol until anesthetized, then 2.5%/volthroughout the procedure. Control animals received the same volume ofvehicle (100% ethanol) alone. Pancreatitis pain was assessed bydetermination of abdominal sensitivity to probing the abdomen of ratswith a calibrated von Frey filament (4 g). Rats were allowed toacclimate in suspended wire-mesh cages for 30 min before testing. Aresponse was indicated by the sharp withdrawal of the abdomen, lickingof abdominal area, or whole body withdrawal. A single trial consisted of10 applications of von Frey filament applied once every 10 s to allowthe animal to cease any response and return to a relatively inactiveposition. The mean occurrence of withdrawal events in each trial isexpressed as the number of responses to 10 applications. Rats withoutinflammation of the pancreas typically display withdrawal frequencies toprobing with von Frey filament of 0-1. The animals were allowed torecover for 6 days after DBTC administration prior to anypharmacological manipulations Animal not demonstrating sufficientabdominal hypersensitivity (i.e., rats with less than 5 positiveresponses out of a possible 10) were excluded from the study.

The number of positive responses, following abdominal probing (out of apossible 10), were recorded at each time point. Data are presented asaverage number of withdrawals (±SEM) for each dosing group at eachcorresponding time point. For dose-response analysis, raw data wereconverted to % maximum possible effect (% MPE) using the formula: %MPE=((test score−post DBTC score)/(pre DBTC score−post DBTC score))*100.Raw data were analyzed using a two-way repeated measures ANOVA followedby Bonferroni post-tests. The dose which elicited 50% attenuation ofhypersensitivity (ED₅₀) was determined using linear regression analysis.Compounds were administered by the intraperitoneal route. FIG. 9summarizes the results of these experiments. The calculated ED₅₀ forcompound (2) in this model was 0.03 mg/kg (0.006-0.14; 95% confidenceinterval).

To determine if the efficacy of Compound (2) (1 mg/kg) is mediated viaactivation of peripheral kappa opioid receptors, groups of eight ratswere pretreated with either the selective kappa opioid receptorantagonist nor-BNI (1 mg/kg), or with a non-selective opioid receptorantagonist, naloxone methiodide (10 mg/kg), which does not cross theblood-brain barrier, prior to treatment with compound (2). FIG. 10summarizes the results of these studies.

Example 122 Pruritus Model in Mice

Groups of 10 (and in one case, 11) male Swiss Webster mice (25-30 g)were used. Each animal was weighed and allowed to acclimate for at leastone hour in individual, rectangular observation boxes. The tails of micewere immersed for 30 seconds in warm water to dilate tail veins and theanimals then received an intravenous injection of either vehicle(saline) or compound (2) (0.01, 0.03, 0.10 and 0.30 mg of free base/kg).Fifteen minutes later, each mouse was given either GNTI dihydrochloride(Tocris) (0.30 mg/kg; 0.25 ml/25 g) or compound 48/80 (Sigma) (50 μg in0.10 ml saline) subcutaneously behind the neck. The animals were thenobserved in pairs (occasionally in threes) and the number of hind legscratching movements directed at the neck was counted for 30 minutes.The mean percent inhibition of scratching caused by compound (2) wasplotted and the dose associated with 50% inhibition was obtained bylinear regression analysis (PharmProTools). Table VII summarizes theresults of these experiments.

TABLE VII Effects of Compound (2) on Pruritus Induced by Either Compound48/80 or GNTI in Mice. Compound 48/80 Model GNTI Model ED₅₀(mg/kg, iv,ED₅₀(mg/kg, iv, Compound # 15 min post-dose) 15 min post-dose) (2) 0.08(0.04-0.2) 0.05 (0.02-0.1)

Example 123 CFA Model for Inflammatory Pain in Rats

Induction of inflammatory pain: Local inflammation was induced in maleSprague-Dawley rats (weighing 200-250 grams) by 50 μL complete Freund'sadjuvant (CFA; Sigma, Mycobacterium tuberculosis 1 mg/mL) injectedsubcutaneously into the plantar surface of the left hind paw. Testcompounds were administered and evaluated for efficacy 24 hr followingCFA administration.

Assessment of inflammatory pain: Paw withdrawal thresholds (PWTs) inresponse to a noxious mechanical stimulus were measured using theRandall-Selitto paw pressure apparatus (Ugo Basile). This apparatusgenerates a linearly increasing mechanical force. The stimulus isapplied to the dorsal surface of the hind paws by a dome-shaped plastictip placed between the 3rd and 4th metatarsus. To avoid tissue damage, acut-off pressure was set at 390 grams. PWTs were defined as the force ingrams at the first obvious pain behavior, which includes paw withdrawal,struggle, and/or vocalization. Rats were tested prior to, and 24 hrfollowing, CFA administration. After about 30 minutes, vehicle or testcompound was administered and PWT tests were carried out over a periodof 2 hours. See Table VIII (below).

Animals not demonstrating sufficient mechanical hypersensitivityfollowing CFA administration (i.e., rats with PWTs>150 g) were excludedfrom the study. All behavioral assessments were carried out underblinded conditions. Intravenous administration of Compound (2) produceda dose- and time-dependent reversal of mechanical hypersensitivityproduced by unilateral CFA-induced inflammatory pain.

The two-way repeated measures ANOVA revealed significant main effects ofDose and Time; F(4,217)=152.1, p<0.0001 and F(6,217)=60.8, p<0.0001,respectively. Further the Dose×Time interaction was statisticallysignificant; F(24,217)=16.7, p<0.0001. Compound (2) produced a markedincrease in PWTs of the injured hind paw, elevating thresholds abovepre-CFA levels. Further, the anti-hyperalgesic actions of Compound (2)persisted for 90 min at the 0.1 mg/kg and at least 120 min at doses of0.3 mg/kg and above. The calculated ED₅₀ for Compound (2) was 0.09 mg/kg(0.07-0.12; 95% CI) 30 min following intravenous administration.

TABLE VIII Dose-dependent attenuation of CFA-induced mechanicalhyperalgesia by intravenous compound (2) Time Vehicle Compd (2) Compd(2) Compd (2) Compd (2) (min) alone (0.03 mg/kg) (0.1 mg/kg) (0.3 mg/kg)(1 mg/kg) Naive 249 ± 4 243 ± 11 239 ± 8 215 ± 6 216 ± 8 CFA 112 ± 6 119± 9 122 ± 8 115 ± 6 116 ± 6 15 107 ± 6 130 ± 5 182 ± 20 *** 261 ± 17 ***244 ± 22 *** 30 111 ± 5 141 ± 9 197 ± 8 *** 328 ± 24 *** 328 ± 19 *** 60102 ± 8 118 ± 9 178 ± 8 *** 212 ± 6 *** 275 ± 10 *** 90 105 ± 4 100 ± 9171 ± 11 *** 219 ± 15 *** 239 ± 15 *** 120  110 ± 5 110 ± 14 118 ± 6 241± 15 *** 263 ± 8 *** Values are reported as mean PWTs ± SEM; *** denotesp < 0.001 vs. Vehicle (Bonferroni tests)

Example 124 Carrageenan Model for Inflammation in Rats

Induction of inflammation: Hindpaw swelling was induced by a singleintraplantar injection of λ-carrageenan (0.1 mL, 2% w/v in deionizedwater).

Assessments of hind paw inflammatory swelling (edema): Paw volumemeasurements were obtained prior to, and 3 hours following, unilateralcarrageenan administration using a plethysmometer (Ugo Basile). Vehicle(saline) was administered in one hindpaw and test compound wasadministered in the lateral hindpaw 30 minutes prior to carrageenaninjection. Rats were gently restrained having their ankles stabilized.Once stabilized, a hind paw was submerged (up to the ankle joint) forapproximately 1 second into a known volume of buffer and the total fluiddisplaced was recorded. This procedure was repeated for both hind paws.

Intravenous administration of Compound (2) also resulted in asignificant, dose-related suppression of carrageenan-induced pawswelling. Thirty minutes prior to carrageenan administration, Compound(2) was administered intravenously, then paw volumes were assessed 3.5hr post treatment (i.e., 3 hr post-carrageenan injection). Compound (2)produced a marked inhibition of paw swelling, achieving an equivalenteffect to ibuprofen (Table 3). The minimum effective anti-inflammatorydose of Compound (2) determined in this model was 0.3 mg/kg.

TABLE X Intravenous Compound (2) dose-dependently attenuatescarrageenan-induced hind paw swelling Vehicle Ibuprofen Compd (2) Compd(2) Compd (2) alone (100 mg/kg) (0.1 mg/kg) (0.3 mg/kg) (1 mg/kg) 1.4 ±0.1 0.8 ± 0.1* 1.3 ± 0.2 0.9 ± 0.1* 0.9 ± 0.1* Data are expressed aschange in paw volume (inflamed-non-inflamed) in mL ± SEM. *denotes p <0.05 vs. Vehicle (Dunnett's tests)

Example 125 Synoviocyte Division Model of Human Rheumatoid Arthritis

General methods for synoviocyte cell culture are well known in the art.See for example: Seki et al., Arthritis Rheum. 41:1356-1364 (1998) andU.S. Pat. No. 7,244,573 to Carman et al. and U.S. Pat. No. 7,348,162 toSaris et al.

Human synovium was removed from the joint of a rheumatoid arthritissubject, cut into small pieces and then digested with collagenase whileshaking at 37° C. The resulting cells were washed twice and platedwithout any further isolation procedures.

Two days after plating the cells were replated in 48 well plates in cellculture media containing fetal bovine serum. The cells were incubatedwith test or reference compound after a further 2 days of plating.Following Giemsa staining at two weeks after incubation with test orreference compound, the synoviocyte cell numbers were counted directlyin each well by microscopy. FIG. 11 shows the inhibition of synoviocytecell proliferation over a range of concentrations of Compound (2) from0.01 nM to 50 nM. The apparent EC₅₀ for inhibition by Compound (2) isapproximately 0.16 nM. For comparison, parallel assays with theglucocorticosteroid, Budesonide used as an anti-inflammatory in thetreatment of rheumatoid arthritis, exhibited an EC₅₀ of approximately0.21 nM for inhibition of synoviocyte proliferation.

Example 126 Effect of Compound (2) in the Rat Monoiodoacetate Model ofOsteoarthritis Knee Pain

Injection of monoiodoacetate (MIA) into the knee joints of rats evokes apainful response that measured as a weight bearing difference betweenthe MIA-injected knees and saline-injected knees and is used as a modelfor human osteoarthritis (OA).

The right knees of approximately 8 week old male Lewis rats wereinjected with 0.3 mg MIA in 50 uL of saline through the patella ligamentinto the synovial joint space and left knee swere injected with 50 uL ofsaline as control. MIA injected rats were dosed sub-cutaneously on day12 after MIA injection with either vehicle (sterile water forinjection), or compound (2) at 0.1 mg/kg, 0.3 mg/kg, or 1 mg/kg in avolume of 1 mL/kg (six rats per group). Incapacitance testing wasmeasured as the difference in hind paw weight bearing between the MIAand saline-injected knees. Three separate measurements each over asecond were averaged. Data is shown as mean with standard error aboutthe mean. Groups were compared using Dunnet's test and data with a Pvalue of less than 0.05 was accepted as significant.

Results: Subcutaneous injection of vehicle (water) alone gave thebase-line difference between right and left hind paw weight bearingmeasured on day 19 post MIA-injection, was 22 g. Animals receiving 0.1mg/kg compound (2) exhibited a mean difference between right and lefthind paw weight bearing of 15 g with a P<0.0002 (n=6) and thosereceiving 0.3 mg/kg compound (2) exhibited a mean difference betweenright and left hind paw weight bearing of 13 g with a P<0.0001 (n=4).

Example 127 Rat Dural Plasma Protein Extravasation as a Migraine Model

Plasma protein extravasation from the dura of laboratory animalsfollowing electrical stimulation of the trigeminal ganglion is used as amodel to test migraine therapies. Protein extravasation from the dura ofelectrically stimulated rats was used to test compound (2) as atreatment candidate for migraine. Sumatriptan was used as a positivecontrol. Male Sprague-Dawley rats (250-350 g) were injectedsubcutaneously with water, sumatriptan or compound (2) at 30, 3, 1 and0.3 mg/kg, each in 1 mL/kg approximnately 20 mins Before beinganesthetized with Nembutal (60 mg/kg, ip) and placed in a stereotacticframe (David Kopf Instruments) with the incisor bar set at −2.5 mm Amidline sagital scalp incision was made and two pairs of bilateral holeswere drilled throught he skull (3.2 mm posterioirly, 1.8 and 3.8 mmlaterally, coordinates being referenced to bregma. Pairs of stainlesssteel electrodes (Rhodes Med. Systems Inc.) insulated except at the tipwere lowered through the holes in both hemispheres to a depth of 9.2 mmbelow the dura. After 28 mins After dosing a solution of fluorosceinisothiocyanate-labelled bovine serum albumin (FITC-BSA, Sigma A977 20mg/kg) was injected into the femoral vein. After 2 mins The lefttrigeminal ganglion was stimulated, 1.0 mA, 5 Hz, 5 mins duration with aModel S48 Grass Instrument Stimulator. Rats were then exsanguinated with40 mL saline. The top of the skull was removed and the dura collected.Membrane samples were removed from both hemispheres and rinsed withwater and spread on microscope slides. The slides were dried at roomtemperature and covered with cover slips and a 70% glycerol/watersolution.

Slides were quantified with a fluorescence microscope (Zeiss) equippedwith a grating monochromator and a spectrophotometer for quantifyingFTIC-BSA in each dura sample. Data were collected on a personal computerfrom the motorized stage reading fluorescence measurements at 25 pointsin 500 μm steps on each sample. The extravasation ratio (fluorescencefrom the stimulated side compared to the unstimulated side) wascalculated. Vehicle alone or ineffective treatments resulted in anextravasation ratio of approximately 2, whereas totally effectivetreatments resulted in an extravasation ratio of approximately 1.Results wee expressed as mean values with standard errors of the mean.Statistical evaluations using ANOVA were followed by comparison tocontrol groups by Dunnet's test. P<0.05 was accepted as significant.

Vehicle alone gave an extravasation ratio of approximately 1.9 andsumatriptan gave an extravasation ratio of approximately 1.1; Compound(2) at 0.3, 1, 3 and 30 mg/kg yielded extravasation ratios ofapproximately 1.8 (P>0.05), 1.6 (P>0.05), 1.0 (P<0.05) and 1.2 (P<0.05)respectively.

Example 128 Effect of Compound (2) on Human Macrophage CytokineProduction

The anti-inflammatory efficacy of a candidate molecule was measured asreduction of cytokine production after lipopolysaccharide (LPS) andIFN-γ induction of human monocytes. The cytokines monitored were IL-1β,IL-8, TNFα and GM-CSF. The anti-inflammatory cytokine, IL-10 was used aspositive control.

Monocyte isolation and incubation: Monocytes were isolated from anindividual female Caucasian subject by density gradient centrifugationin percoll and plated in 48-well plates at 2×10⁵ cells/ml/well. Cellswere cultured in Iscove's medium (invitrogen, UK) supplemented withpenicillin (100 units/ml)/streptomycin (100 ug/ml), nonessential; 1×amino acids, 1 mM sodium pyruvate, 25 mM HEPES, 10% FBS (Invitrogen,UK), 2 ng/ml GM-CSF (PeproTech, UK). After 7 days in culture cells wereexposed to on of the following: (i) 0.1 nM, 1 nM, 3 nM, 10 nM or 30 nMcompound (2); (ii) 10 ng/ml IL-10; for 4 hours prior to addition of 1ng/ml LPS+10 ng/ml IFN-γ (PeproTech, UK) for 18 hours. Assays wereperformed in triplicate.

Analysis of cytokines: Bead-based assays for each cytokine were analysedon a Luminex-100 according to the manufacturer's instructions.Statistical comparisons were by ANOVA with Dunnet's and Bonferroni'spost analysis test for concentration-response curves and effects ofnor-BNI respectively, using Graph Pad Prism software. Statisticalsignificance was accepted at the 0.05 level.

TNF: Percent of Untreated Level (Approximate)

Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 TNFα % 100 95 35 55 70 55 4520

IL-1β: Percent of Untreated Level (Approximate)

Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 IL-1β % 100 80 10 5 25 −10 −2540

IL-8: Percent of Untreated Level (Approximate)

Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 IL-8 % 100 90 75 70 60 45 4025

GM-CSF: Percent of Untreated Level (Approximate)

Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 GM-CSF % 100 20 40 −30 −5 −55−60 −40

Example 129 Effect of Compound (2) on Human Synoviocyte CytokineProduction

Synoviocytes were prepared as described in Example 125 above, two dayslater the cells were replated in tissue culture treated 96-well plates.After a further two days cells were incubated with compound (2) at 0.1,0.3, 1, 3, 10 and 30 nM at 4 hours before stimulus. Cytokine productionwas stimulated with IFNγ (10 ng/mL, PeproTech, UK) and Mab CD40 (1μg/mL, eBiosciences, UK) for 4 hours before assaying MMPs and 48 hoursbefore TNFα was assayed. Cells stimulated with IFNγ and Mab CD40 wereused as standards. Budesonide (1 μM) was added 1 hour before stimulus aspositive control. TNFα and MMP-1 and MMP-3 assays were analysed on aLuminex 100 according to the manufacturer's instructions. Statisticalcomparisons were by ANOVA with Dunnet's test for multiple comparisonsusing Graph Pad Prism ver. 5 software. Statistical significance wasaccepted at the P<0.05 level.

TNF: Percent of Untreated Level (Approximate)

Compound (2) nM 0 0.1 0.3 1 3 10 30 Budesonide TNFα % 100 20 −25 −25 −20−30 −45 −30

MMP-1: Percent of Untreated Level (Approximate)

Compound (2) nM 0 0.1 0.3 1 3 10 30 Budesonide MMP-1 % 100 10 −40 −250−220 −120 −200 −240

MMP-3: Percent of Untreated Level (Approximate)

Compound (2) nM 0 0.1 0.3 1 3 10 30 IL-10 MMP-3 % 100 −25 0 −15 −15 −20−30 −35

Elevated levels of the pro-inflammatory cytokines TNFα, IL1 β, IL-6, IL8and IL-10 have been linked to propensity for glucose intolerance,insulin resistance, diabetes and obesity. Development of theseconditions can be prevented or at least inhibited by the administrationof the synthetic peptide amides of the present invention. Treatmentsaccording to the present invention are useful as adjunct therapies forthese conditions.

The specifications of each of the U.S. patents and published patentapplications, and the texts of the literature references cited in thisspecification are herein incorporated by reference in their entireties.In the event that any definition or description contained found in oneor more of these references is in conflict with the correspondingdefinition or description herein, then the definition or descriptiondisclosed herein is intended.

The examples provided herein are for illustration purposes only and arenot intended to limit the scope of the invention, the full breadth ofwhich will be readily recognized by those of skill in the art.

1. A method of prophylaxis or treatment of a kappa opioidreceptor-associated disease or condition in a mammal, the methodcomprising administering to the mammal a composition comprising aneffective amount of a synthetic peptide amide of the formula:

or a stereoisomer, mixture of stereoisomers, prodrug, pharmaceuticallyacceptable salt, hydrate, acid salt hydrate, N-oxide or isomorphiccrystalline form thereof, wherein each Xaa₁ is independently selectedfrom the group consisting of (A)(A′)D-Phe, (A)(A′)(α-Me)D-Phe, D-Tyr,D-Tic, D-tert-leucine, D-neopentylglycine, D-phenylglycine,D-homophenylalanine, β-(E)D-Ala and tert-butyl-D-Gly, wherein each (A)and each (A′) are phenyl ring substituents independently selected fromthe group consisting of —H, —F, —Cl, —NO₂, —CH₃, —CF₃, —CN, —CONH₂, andwherein each (E) is independently selected from the group consisting oftert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, furyl,pyridyl, thienyl, thiazolyl and benzothienyl; each Xaa₂ is independentlyselected from the group consisting of (A)(A′)D-Phe, (A)(A′)(α-Me)D-Phe,D-1Nal, D-2Nal, D-Tyr, (E)D-Ala, and D-Trp; each Xaa₃ is independentlyselected from the group consisting of D-Nle, D-Phe, (E)D-Ala, D-Leu,(α-Me)D-Leu, D-Hle, D-Val, and D-Met; each Xaa₄ is independentlyselected from the group consisting of (B)₂D-Arg, (B)₂D-Nar, (B)₂D-Har,ζ-(B)D-Hlys, D-Dap, ε-(B)D-Lys, ε-(B)₂-D-Lys, D-Amf, amidino-D-Amf,γ-(B)₂D-Dbu, δ-(B)₂α-(B′)D-Orn, D-2-amino-3(4-piperidyl)propionic acid,D-2-amino-3(2-aminopyrrolidyl)propionic acid,D-α-amino-β-amidino-propionic acid, α-amino-4-piperidineacetic acid,cis-α,4-diaminocyclohexane acetic acid,trans-α,4-diaminocyclohexaneacetic acid,cis-α-amino-4-methyl-aminocyclo-hexane acetic acid,trans-α-amino-4-methylaminocyclohexane acetic acid,α-amino-1-amidino-4-piperidineacetic acid,cis-α-amino-4-guanidino-cyclohexane acetic acid, andtrans-α-amino-4-guanidinocyclohexane acetic acid, wherein each (B) isindependently selected from the group consisting of —H and C₁-C₄ alkyl,and (B′) is —H or (α-Me), and p is zero or 1; G is (i)

wherein q, r and s are each independently zero or 1; p and t are eachindependently zero or 1; provided that at least one of q, r, s and t is1; L is a linker selected from the group consisting of ε-D-Lys, ε-Lys,δ-D-Orn, δ-Orn, γ-aminobutyric acid, 8-aminooctanoic acid,11-amino-undecanoic acid, 8-amino-3,6-dioxaoctanoic acid,4-amino-4-carboxylic piperidine and bis(D-Lys-Gly)Lactam; or G is (ii)

wherein p is 1; and the moiety

is an optionally substituted 4 to 8-membered heterocyclic ring moietywherein Y is C or N and Z is C, N, O, S, SO, or SO₂; provided that whensuch ring moiety is a 6-, 7- or 8-membered ring, Y and Z are separatedby at least two ring atoms; and provided further that when such ringmoiety is aromatic, then Y is carbon; wherein W is selected from thegroup consisting of: null, provided that when W is null, Y is N;—NH—(CH₂)_(b)- with b equal to zero, 1, 2, 3, 4, 5, or 6; and—NH—(CH₂)_(c)-O— with c equal to 2, or 3; wherein V is C₁-C₆ alkyl, ande is zero or 1, wherein when e is zero, then V is null and, R₁ and R₂are directly bonded to the same or different ring atoms; wherein (a) R₁is —H, —OH, halo, CF₃, —NH₂, —COOH, C₁-C₆ alkyl, C₁-C₆ alkoxy, amidino,C₁-C₆ alkyl-substituted amidino, aryl, optionally substitutedheterocyclyl, Pro-amide, Pro, Gly, Ala, Val, Leu, Ile, Lys, Arg, Orn,Ser, Thr, CN, CONH₂, COR′, SO₂R′, CONR′R″, NHCOR′, OR′, or SO₂NR′R″;wherein said optionally substituted heterocyclyl is optionally singly ordoubly substituted with substituents independently selected from thegroup consisting of C₁-C₆ alkyl, —C₁-C₆ alkoxy, oxo, —OH, —Cl, —F, —NH₂,—NO₂, —CN, —COOH, and amidino; wherein R′ and R″ are each independently—H, C₁-C₈ alkyl, aryl, or heterocyclyl or R′ and R″ are combined to forma 4- to 8-membered ring, which ring is optionally substituted singly ordoubly with substituents independently selected from the groupconsisting of C₁-C₆ alkyl, —C₁-C₆ alkoxy, —OH, —Cl, —F, —NH₂, —NO₂, —CN,—COOH and amidino; and R₂ is H, amidino, singly or doubly C₁-C₆alkyl-substituted amidino, —CN, —CONH₂, —CONR′R″, —NHCOR′, —SO₂NR′R″, or—COOH; or (b) R₁ and R₂ taken together can form an optionallysubstituted 4- to 9-membered heterocyclic monocyclic or bicyclic ringmoiety which is bonded to a single ring atom of the Y and Z-containingring moiety; or (c) R₁ and R₂ taken together with a single ring atom ofthe Y and Z-containing ring moiety can form an optionally substituted 4-to 8-membered heterocyclic ring moiety to form a spiro structure; or (d)R₁ and R₂ taken together with two or more adjacent ring atoms of the Yand Z-containing ring moiety can form an optionally substituted 4- to9-membered heterocyclic monocyclic or bicyclic ring moiety fused to theY and Z-containing ring moiety; wherein each of said optionallysubstituted 4-, 5-, 6-, 7-, 8-, and 9-membered heterocyclic ringmoieties comprising R₁ and R₂ is optionally singly or doubly substitutedwith substituents independently selected from the group consisting ofC₁-C₆ alkyl, —C₁-C₆ alkoxy, optionally substituted phenyl, oxo, —OH,—Cl, —F, —NH₂, —NO₂, —CN, —COOH, and amidino; provided that when the Yand Z-containing ring moiety is a six or seven membered ring comprisinga single ring heteroatom and wherein such heteroatom is N, and e iszero, then R₁ is not —OH, and R₁ and R₂ are not both —H; providedfurther that when the Y and Z-containing ring moiety is a six memberedring comprising two ring heteroatoms, both Y and Z are N, W is null, and—V_(e)(R₁)(R₂) is attached to Z, then —V_(e)(R₁)(R₂) is selected fromthe group consisting of amidino, C₁-C₆ alkyl-substituted amidino,dihydroimidazole, —CH₂COOH, and —H₂C(O)NH₂; and lastly, provided that ifthe Y and Z-containing ring moiety is a six membered ring comprising anS or O ring heteroatom, or if the Y and Z-containing ring moiety is asix membered ring comprising two ring heteroatoms, wherein both Y and Zare N and W is null, or if the Y and Z-containing ring moiety is a sixmembered aromatic ring comprising a single ring heteroatom, whichheteroatom is N, then, when e is zero, R₁ and R₂ are not both —H.
 2. Themethod according to claim 1, wherein the kappa opioidreceptor-associated disease or condition is an inflammatory disease orcondition.
 3. The method according to claim 2, wherein the inflammatorydisease or condition is selected from the group consisting ofcardiovascular inflammation, neurological inflammation, skeletalinflammation, muscular inflammation, gastrointestinal inflammation,ocular inflammation, otic inflammation, inflammation due to insect bitesand inflammation due to wound healing.
 4. The method according to claim3, wherein the cardiovascular inflammation is due to atherosclerosis,ischemia, restenosis or vasculitis.
 5. The method according to claim 2,wherein the inflammatory disease or condition is selected from the groupconsisting of asthma, Sjogren's syndrome, pulmonary inflammation,chronic airway inflammation and chronic obstructive pulmonary disease(COPD).
 6. The method according to claim 2, wherein the inflammatorydisease or condition is selected from the group consisting of allergy,psoriasis, psoriatic arthritis, eczema, scleroderma, atopic dermatitisand systemic lupus erythematosus.
 7. The method according to claim 2,wherein the inflammatory disease or condition is selected from the groupconsisting of arthritis, synovitis, osteomyelitis, rheumatoid arthritis,osteoarthritis and ankylosing spondylitis.
 8. The method according toclaim 2, wherein the inflammatory disease or condition is selected fromthe group consisting of septicemia and septic shock.
 9. The methodaccording to claim 2, wherein the inflammatory disease or condition isselected from the group consisting of diabetes, glucose intolerance,insulin resistance and obesity.
 10. The method according to claim 2,wherein the inflammatory disease or condition is selected from the groupconsisting of colitis, ulcerative colitis, Crohn's disease, IBD(inflammatory bowel disease) and IBS (Irritable bowel syndrome).
 11. Themethod according to claim 2, wherein the inflammatory disease orcondition is due to tumor proliferation, tumor metastasis ortransplantation rejection.
 12. The method according to claim 1, whereinthe mammal is a human.
 13. The method according to claim 12, wherein thedisease or condition is migraine.
 14. The method according to claim 13,wherein the synthetic peptide amide is selected from the groupconsisting of compounds (1)-(105).
 15. A method according to claim 1,wherein the inflammatory disease or condition is associated withelevated levels of a proinflammatory cytokine.
 16. The method accordingto claim 15, wherein the proinflammatory cytokine is selected from thegroup consisting of TNF-α, IL-1β, IL-6, MMP-1 and MMP-3.
 17. A method ofprophylaxis or treatment of an inflammatory disease or condition in amammal, the method comprising administering to the mammal a compositioncomprising an effective amount of a synthetic peptide amide having astructure selected from the group consisting of compounds (1)-(105). 18.The method according to claim 17, wherein the mammal is a human.
 19. Themethod according to claim 17, wherein the the inflammatory disease orcondition is associated with elevated levels of a proinflammatorycytokine.
 20. The method according to claim 17, wherein theproinflammatory cytokine is selected from the group consisting of TNF-α,IL-1β, IL-6, MMP-1 and MMP-3.